Isotopically coded affinity markers 3

ABSTRACT

This application relates to isotopically labeled affinity markers of the formula (II) 
     
       
         
         
             
             
         
       
         
         
           
             for mass spectrometric analysis of proteins. In formula (II), the groups A, PRG, S, Z, L&#39;, Z&#39;, R, R&#39;, k, l, m, and n are as defined in the claims. The application also provides a process for preparing these materials, a method for analyzing proteins using such materials, and a kit containing one or more of these materials.

The invention relates to novel, isotope-coded affinity tags for themass-spectrometric analysis of proteins, and to their preparation anduse.

Proteomics technology opens up the possibility of identifying novelbiological targets and tags by means of analyzing biological systems atthe protein level. It is known that only a certain proportion of all thepossible proteins encoded in the genome is being expressed at any giventime, with, for example, tissue type, state of development, activationof receptors or cellular interactions influencing the pattern and ratesof expression. In order to detect differences in the expression ofproteins in healthy or diseased tissue, it is possible to make use of avariety of comparative methods for analyzing protein expression patterns((a) S. P. Gygi et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 9390;(b) D. R. Goodlett et al., Proteome Protein Anal. 2000, 3; (c) S. P.Gygi et al., Curr. Opin. Biotechnol., 2000, 11, 396).

The mass-spectrometric detection of proteins is a powerful method inthis connection. When affinity tags which have been isotope-codeddifferentially (ICAT®=isotope coded affinity tags) and tandem massspectrometry are used, this method can be enlisted for quantitativelyanalyzing complex protein mixtures ((a) S. P. Gygi et al., NatureBiotechnology, 1999, 17, 994; (b) R. H. Aebersold et al., WO 00/11208).The method is based on each of two or more protein mixtures, which areto be compared and which have been obtained in different cell states,being reacted with an affinity tag of a different isotope coding. Afterthat, the protein mixes are combined, where appropriate fractionated ortreated proteolytically and purified by affinity chromatography. Afterthe bound fragments have been eluted, the eluates are analyzed by acombination of liquid chromatography and mass spectrometry (LC-MS).Pairs or groups of peptides which are labeled with affinity tags whichonly differ in the isotope coding are chemically identical and areeluted virtually simultaneously in the HPLC; however, they differ in themass spectrometer by the respective molecular weight differences due tothe affinity labels having different isotope patterns. Relative proteinconcentrations can be obtained directly by carrying out measurements ofthe peak areas. Suitable affinity tags are conjugates composed ofaffinity ligands which are linked covalently to protein-reactive groupsby way of bridge members. In connection with this, different isotopesare incorporated into the bridge members. The method was described usingaffinity tags in which hydrogen atoms were replaced with deuterium atoms(¹H/²D isotope coding).

The method using ¹H/²D isotope-coded affinity tags which is described inthe prior art suffers from a variety of disadvantages, in particular anisotope effect of the differently labeled, but otherwise identical,peptide fragments in the LC, inadequate stability of the affinity tagsin general and especially in LC-MS/MS, a lack of efficiency as regardsthe avidin monomer-based affinity chromatography, and a lack offlexibility in regard to the incorporation of the isotope labels.

The object of the present invention was to make available improvedisotope-coded affinity tags.

The invention relates to organic compounds which are suitable for use asaffinity tags for the mass-spectrometric analysis of proteins, of theformula (I),A-L-PRG   (I)in which

-   -   A is an affinity ligand residue or a solid phase,    -   PRG is a protein-reactive group, and    -   L is a linker which covalently links A and PRG,    -   where the linker L contains a group of the formula (I′)

in which

-   -   Z is the amino acid residue NH—CHR^(x)—(CH₂)_(x)—CO, with the        amino acid side chain being R^(x) and the number x being        selected from the range of 0 to 5,    -   L′ is a bridge which makes possible or facilitates a covalent        linkage of two piperazine residues or, in the case of 1≧2, are        identical or different bridges of this nature,    -   R and R′ are in each case an α-amino acid side chain on a        piperazine ring, which side chains are independent of each other        and independent of other R and R′ on other piperazine rings        belonging to the same running variable or the other of the two        running variables 1 and m,    -   Z′ is the amino acid residue CO—(CH₂)_(y)—CHR^(y)—NH, with the        amino acid side chain being R^(y) and the number y being        selected from the range of 0 to 5, with Z′ differing from Z in        the different orientation in regard to the terminal CO and NH        groups, and    -   k, l, m and n are, independently of each other, in each case a        number from 0 to 10, with the sum k+l+m+n being at least 1 and        at most 40,        or the salts thereof.

As compared with the prior art, the affinity tags in accordance with theinvention exhibit the following advantages, in particular:

deuterium, ¹³C-labelled or ¹³C-labelled and ¹⁵N-labelled glycinebuilding blocks, or other correspondingly labeled amino acid buildingblocks, are inexpensive starting materials which make it possible toconstruct the isotope-labeled affinity tags in a flexible manner. Morethan twenty ¹³C labels and, in addition, up to ten ¹⁵N isotopes can bereadily introduced into the affinity tag which is described in theformula (I). In contrast to the previously described affinity tagshaving a markedly smaller mass difference (ΔM=8), it is also possible,in this way, to analyze in parallel several proteome samples byrespectively modifying with affinity tags having various massdifferences. Preference is given to the embodiment using up to 4differently labeled affinity tags, which permit the simultaneousanalysis and relative quantification of up to 4 complex proteomesamples.

The modular construction of the affinity tags permits a flexiblecombination of the individual building blocks, which combination matchesthe requirements of the operational program.

The affinity tags which are described can be equipped with anacid-labile predetermined breaking point which enables the peptidefragments to be decomplexed by means of a substantially more efficientaffinity chromatography, which is based, for example, on streptavidin oroligomeric avidin in the case of the biotin-modified peptide fragments,or by means of reversible binding to a solid phase. Furthermore, thetags which remain on the peptide fragments following acid cleavage havea low molecular weight and a high isotope density.

The manipulation of the affinity tags is improved as the result of animprovement in solubility, as the result of being in a crystalline oramorphous state and as the result of an increase in stability.

The invention furthermore relates to the use of one or more compoundsaccording to the invention, which are isotope-labeled differently, asreagents for the mass-spectrometric analysis of proteins, in particularfor identifying one or more proteins or protein functions in one or moreprotein-containing samples, and for determining the relative expressionlevels of one or more proteins in one or more protein-containingsamples.

The invention furthermore relates to a kit for the mass-spectrometricanalysis of proteins, which kit contains, as reagents, one or morecompounds according to the invention which are isotope-labeleddifferently.

An affinity ligand A is used for selectively enriching samples by meansof affinity chromatography. The affinity columns are provided with thecorresponding reactants which are complementary to the affinity ligands,and which enter into covalent or noncovalent bonds with the affinityligands. An example of a suitable affinity ligand is biotin or a biotinderivative, which enters into strong, noncovalent bonds with thecomplementary peptides avidin or spectravidin. In this way, it ispossible to use affinity chromatography to selectively isolate samplesto be investigated from sample mixtures. In the same sense, it is alsopossible for example, to use carbohydrate residues, which are able toenter into noncovalent interactions with fixed lectins, for example, asaffinity ligands. It is furthermore possible to use the interaction ofhaptens with antibodies, or the interaction of transition metals withcorresponding ligands, as complexing agents, or other systems whichinteract with each other, in the same sense.

Alternatively, the selective enrichment can also be achieved by means ofselective, reversible binding to an appropriately functionalized solidphase A. Examples of suitable solid phases are amino-functionalizedresins based on silica gel and, furthermore, those known from thepeptide syntheses carried out as solid phase syntheses, such as tritylresin, Sasrin resin, which is based on benzyl alcohol supportation, Wangresin, which is based on benzyl alcohol supportation, Wang polystyreneresin, Rink amide MBHA resin or TCP (trityl chloride polystyrene) resin(in the formulae which are depicted, the encircled P in each caserepresents the resin residue):

A polymeric support, in particular a modified natural or syntheticresin, for example a resin based on silica gel or polyethylene glycol,which possesses functional groups, such as hydroxyl, carboxyl and aminogroups, in particular amino groups, which are suitable for binding onthe linker L, is preferred as the solid phase A. An amino-functionalizedresin based on silica gel, for example an aminopropyl silica gel as ismarketed, for example, by Aldrich under the number 36425-8, isparticularly preferred as the solid phase A.

Protein-reactive groups, PRG, are used for selectively labeling theproteins at selected functional groups. PRGs have a specific reactivityfor terminal functional groups in proteins. Examples of amino acidswhich, as elements or proteins, are frequently used for selectivelabeling, are mercaptoaminomonocarboxylic acids, such as cysteine,diaminomonocarboxylic acids, such as lysine or arginine, ormonoaminodicarboxylic acids, such as aspartic acid or glutamic acid.Furthermore, protein-reactive groups can also be phosphate-reactivegroups, such as metal chelates, and also aldehyde-reactive andketone-reactive groups, such as semicarbazones or else amines,accompanied by subsequent treatment with sodium borohydride or sodiumcyanoborohydride. Protein-reactive groups can also be groups which,following a selective protein derivatization, such as a cyanogen bromidecleavage, or an elimination of phosphate groups, etc., react with thereaction products.

Z and Z′ are residues of identical or different amino acids. Preferenceis given to residues of L-α-amino acids, in particular the 20 naturalproteinogenic amino acids (x=0 or y=0), for example glycine residues,and residues of ω-amino acids, such as NH—(CH₂)₂—CO and CO—(CH₂)₂—NH. Inthis connection, the amino acids can, where appropriate, be in the D, Lor racemic form.

Preferred compounds according to the invention of the formula (I)possess, in the linker L, as the group of the formula (I′), a group ofthe formula (I″) (all the R and R′ in formula (I′) are hydrogens):

Compounds according to the invention of the formula (I) which arelikewise preferred possess, in the linker L, a group of the formula(I′), in particular a group of the formula (I″), in which Z is theglycine residue NH—CH₂—CO or Z′ is the glycine residue CO—CH₂—NH, inparticular in which Z is the glycine residue NH—CH₂—CO and Z′ is theglycine residue CO—CH₂—NH.

In another embodiment, preference is given to linkers L which possess apredetermined breaking point S, such as an acid-labile functionality,which, under particular conditions, for example under the influence ofacid, guarantee cleavage of the affinity tag in order, in this way, forexample, to facilitate release from the affinity column or to reduce thesize of the residue remaining on the peptide or to make the operationalprocedures as a whole more efficient. Instead of using an acid-labilepredetermined breaking point, it is also possible to bring about thecleavage of the linkers L in another manner, for example by chemicalcleavage of, inter alia, silyl ethers, esters, carbamates, thioesters,acetals, disulfides or Schiff's bases, and, furthermore, by means ofphotochemical cleavage or by means of the enzymic cleavage of esters,amides, nucleotides or glycosides or by means of thermal cleavage, forexample of interacting nucleic acid strands.

In order to improve the solubility of the affinity tags according to theinvention, it is possible to prepare and employ acid and/or basicfunctional groups which are present in the form of their salts,preferably their hydrochlorides, acetates, trifluoroacetates, alkalimetal salts or ammonium salts.

In one particular embodiment of the invention, the protein-reactivegroup PRG possesses solubility-improving functional groups.

Preferred compounds according to the invention are those of the formula(I), in particular of the formula (II),

in which one or more of the groups A, PRG, S, Z, L′, Z′ and k, l, m andn, in particular all these groups, are selected in accordance with thefollowing definitions:

-   -   A is the acyl residue of an affinity ligand, for example        biotinyl or a biotin derivative, or is a functional group which        is bound to a polymeric support, for example a support-bound        hydroxyl, carboxyl or amino group, in particular a support-bound        amino group.    -   PRG is the residue of a protein-reactive group which is        characterized by an electrophilic group and a suitable bridge        which makes possible, and facilitates, the bonding of the        electrophilic group to Z′. furthermore, such a group can, by        means of being configured appropriately, also improve the        solubility, for example. A preferred protein-reactive group is        an epoxide, a maleimido group, a halogen or an acrylic residue,        in particular bridged electrophiles such as

—CO—[CH₂]_(r)—Cl—CO—CH═CH₂.

-   -    in which r=1–10,        -   Furthermore, PRG can be another known protein-reactive            group, as were described and summarized, for example,            by W. H. Scouten in Methods in Enzymology, Volume 135,            edited by Klaus Mosbach, AcademicPress Inc. 1987, pp. 30 ff.    -   S is an acid-labile predetermined breaking point, such as

-   -    in which Y is a spacer, preferably having 1–10, in particular        1–5, non-hydrogen atoms, which makes possible or facilitates,        the binding of the aryl residue to the affinity ligands or to        the polymeric support, particularly preferably NH, NH—CH₂,        NH—CH₂—CH₂—NH—CO or CH₂—CO, where Y can be in the ortho, meta or        para position in relation to NH and the para position is        preferred,    -    and in which SK is the side chain residue of an α-amino acid of        the formula SK—CH(NH₂)—COOH, which, in the case of SKs other        than an H atom, can be present in the D, L or racemic form, for        example the side chains of the 20 natural amino acids and their        D forms and racemates.    -   Z is the residue of an amino acid, in particular the residue of        glycine which is not labeled or which can contain ¹³C or ¹⁵N        labels, or a combination of these labels.    -   L′ is a bridge which makes possible, or facilitates, the        covalent linkage of two piperazine residues. L′ is preferably        composed of the building blocks alkylene, in particular        (CH₂)_(s), alkenylene, alkynylene, arylene, CO, CS and NH, in        particular composed of a number of from 2 to 10 of these        building blocks. L′ can contain other amino acid residues, such        as Z and Z′, in particular glycine residues, which can, where        appropriate, contain ¹³C or ¹⁵N isotope labels or a combination        of these labels, and is preferably a bridge selected from CO—CO,        CO—(CH₂)_(s)—CO and also CO-arylene-CO, CO—CH₂—NH—CO—NH—CH₂—CO,        CO—NH—CH₂—CO, CO—CH₂—NH—CO, CO—CH₂—NH—CO—CO—NH—CH₂—CO,        CO—CH₂—NH—CO—(CH₂)_(s)—CO—NH—CH₂—CO,        CO—CH₂—NH—CO—arylene-CO—NH—CH₂—CO,        (CH₂)_(s)—NH—CO—CO—NH—(CH₂)_(s), (CH₂)₃—NH—CO—CO—NH—(CH₂)₃,        (CH₂)_(s)—CO, (CH₂)₂—CO, CO and CS, where s is preferably an        integer between 1 and 6, for example 2, 3, 4 or 5.    -   R and R′ on a piperazine ring are in each case identical amino        acid side chains, in particular hydrogen, and are preferably        identical amino acid side chains, in particular hydrogen, on all        the piperazine rings belonging to one of the two running        variables l and m, and are particularly preferably identical        amino acid side chains, in particular hydrogen, on all the        piperazine rings.    -   Z′ is the residue of an amino acid, in particular the residue of        glycine, which differs from Z in the different orientation in        regard to the terminal CO and NH groups; it may not be labeled        or may contain ¹³C or ¹⁵N labels, or a combination of these        labels.    -   k, l, m and n can, independently of each other, in each case be        numbers between 0 and 10, where the sum of k+l+m+n is preferably        greater than 0 and less than 20, and is particularly preferably        less than 10. m is preferably the number 0 or 1.

Within the context of the present invention, alkyl, alkylene,alkenylene, alkynylene, alkoxy and arylene have the following meaning,unless otherwise specified:

Alkyl per se, and “alk” and “alkyl” in alkylene, alkenylene, alkynyleneand alkoxy, are a linear or branched alkyl radical having as a rule from1 to 6, preferably from 1 to 4, particularly preferably from 1 to 3,carbon atoms, by way of example and preferably methyl, ethyl, n-propyl,isopropyl, tert-butyl, n-pentyl and n-hexyl.

Alkylene, alkenylene and alkynylene are bivalent alkyl groups which, inthe case of alkenylene or alkynylene, possess one, two, three, four,five or more double or triple bonds and correspondingly possess at least2 carbon atoms, for example and preferably methylene, ethylene,ethenylene, ethynylene, n-propylene, isopropylene, n-propenylene,methylethenylene and propynylene.

Alkoxy is, by way of example and preferably, methoxy, ethoxy, n-propoxy,isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.

Arylene is a bivalent mono- to tricyclic aromatic carbocyclic radicalhaving as a rule from 6 to 14 carbon atoms, by way of example andpreferably phenylene, naphthylene and phenanthrenylene.

An amino acid tertiary function which may, where appropriate, be presentin an amino acid side chain R, R′, R^(x), R^(y), or SK can optionally bepresent in free form or protected with a protecting group. In oneparticular embodiment, the side chains, in particular SK, contribute toimproving the solubility of the affinity tags.

The non-isotope-labeled compounds already constitute one isotope coding.For further isotope coding, the compounds according to the invention arepreferably isotope-labeled with at least one carbon atom of the isotope¹³C, in particular from four to 20 ¹³C atoms. The disadvantageousisotope effect which was observed in LC when using ¹H/²D isotope-codedaffinity tags is markedly reduced, and even not evident at all, in thecase of ¹²C/¹³C isotope coding. Alternatively or in addition, it is alsopossible to use the isotopes ²D, ¹⁵N, ¹⁷O, ¹⁸O and/or ³⁴S for thelabeling.

In a particular embodiment of the invention, ¹³C-labeled compounds areadditionally isotope-labeled with at least one nitrogen atom of theisotope ¹⁵N, preferably from one to ten ¹⁵N atoms, in particular fromone to three ¹⁵N atoms.

The isotope labelings are generally carried out in L and/or PRG,preferably in L and, in that case, in particular in the groups Z, L′ andZ′ and/or in the piperazine building blocks.

The compounds according to the invention can, for example, be preparedby initially preparing an intermediate of the formula (III)

which is protected with suitable orthogonal protecting groups SG andSG′, for example with the Boc group and the Fmoc group, in accordancewith formula (III′)

These intermediates are synthesized using classical methods of peptidechemistry as are known to a skilled person and as have been described,for example, in Houben-Weyl; Methoden der Organischen Chemie [Methods oforganic chemistry]; fourth edition; volume XV parts 1 and 2; GeorgThieme Verlag Stuttgart 1974, or in Hans-Dieter Jakubke and HansJeschkeit: Aminosäuren, Peptide, Proteine [Amino acids, peptides,proteins]; Verlag Chemie, Weinheim 1982.

Standard reactions are first of all used to detach the protecting groupSG once again from these intermediates and, after that, whereappropriate, another amino acid derivative, which carries a protectinggroup SG which is identical to, or different from, the detachedprotecting group, in particular a Boc group, on the α-amino function, isattached, with derivatives of the formula (IV) being obtained. Tertiaryfunctions which may be present in SK can optionally be present inprotected form of in free form. Protecting groups which are optionallypresent can be retained permanently or be detached in separateunblocking steps or at the same time as the elimination of one of theterminal protecting groups.

Subsequently, piperidine in DMF can be used to detach the protectinggroup SG′, for example an Fmoc group, from (IV) such that, in the nextstep, the coupling with the derivative of a protein-reactive group orthe activated precursor of the derivative of a protein-reactive group ofthe formulaU-PRG  (V)in which U is a group which permits the linkage of PRG to Z′ or, whereappropriate, to another end group of L, by, for example, becoming aleaving group, can take place. Examples of such groups are, activatedesters, such as N-hydroxysuccinimide esters, or chlorides or groups fromwhich a leaving group can be generated during the coupling.

In a further step, the terminal protecting group SG is then detached,resulting in a conjugate of the formula (VI) being obtained.

In parallel with this, an affinity ligand A-OH or A-NH₂, or an activatedform thereof, such as an activated ester, an acid chloride or the like,or a hydroxyl-functionalized, carboxyl-functionalized oramino-functionalized solid phase A-OH or A-NH₂, or an activated formthereof, is reacted, under suitable coupling conditions, with a compound

which can optionally also carry a protecting group, to give thederivative

Activated carbonic acid derivatives, such as thiophosgene orthiocarbonyl-bisimidazole, are then used to convert the derivative(VIII), where appropriate after prior elimination of an optionallyintroduced protecting group, into a corresponding isothiocyanate, whichis then coupled to (VI) to give the thiourea (IX).

For example, the amino function on resins can initially be reacted withFmoc-protected p-aminobenzoic acid or with Fmoc-protectedp-aminophenylacetic acid; subsequently, the Fmoc group can be detachedand the compound converted into the isothiocyanate using an activatedcarbonic acid derivative; the isothiocyanate can then be converted intothe thiourea.

The invention consequently also relates to a process for preparing acompound as claimed in claim 1, in which process

-   -   i) a protected intermediate of the formula (III)

-   -    in which SG and SG′ are two orthogonal protecting groups,    -    is prepared,    -   ii) the protecting group SG is first of all detached from the        intermediate of the formula (III) and, after that, another amino        acid derivative, which carries a protecting group SG, which is        identical to, or different from, the detached protecting group,        on the α-aminofunction, is attached, with a derivative of the        formula (IV),

-   -    in which SK is the side chain of an amino acid,    -    being obtained,    -   iii) after the protecting group SG′ has been detached from the        derivative of the formula (IV), the latter is reacted with the        derivative of a protein-reactive group or the activated        precursor of the derivative of a protein-reactive group of the        formula (V)        U-PRG  (V)    -    in which U is a group which enables PRG to be linked to Z′ or,        where appropriate, to another end group of L,    -   iv) the terminal protecting group SG is detached, with a        conjugate of the formula (VI)

-   -    being obtained,    -   v) an affinity ligand A-OH or A-NH₂, or a        hydroxyl-functionalized, carboxyl-functionalized or        amino-functionalized solid phase A-OH or A-NH₂, or an activated        form thereof, is reacted with a compound of the formula (VII)

-   -    in which Y is the optionally branched spacer group    -    which can optionally carry a protecting group, to give the        derivative of the formula (VIII)

-   -   vi) the derivative of the formula (VIII) is then converted,        after prior elimination of an optionally introduced protecting        group, into a corresponding isothiocyanate,    -   vii) the isothiocyanate is then coupled to the conjugate of the        formula (VI) to give the thiourea of the formula (IX), and

-   -   viii) in an optional last step, protecting groups which are        still present are eliminated, where appropriate,    -    with it being possible to carry out the consecutive steps v)        and vi) at any arbitrary time prior to step vii).

In all the reaction steps, it is possible to use protecting groups whichcan be reversibly eliminated, as are customary in peptide chemistry. Aprotecting group SG can be retained or detached at the same time as theBoc protecting group or be detached in a separate step. Examples ofsuitable protecting groups are the Boc protecting group, which can becleaved using trifluoroacetic acid, or the Fmoc protecting group, whichcan be cleaved using piperidine or morpholine. Other suitable protectinggroups, and the appropriate methods for introducing and eliminatingthem, have been described, for example, in Jakubke/Jeschkeit;Aminosäuren, Peptide, Proteine [Amino acids, peptides and proteins];Verlag Chemie 1982 or in Houben-Weyl, Methoden der Organischen Chemie[Methods of organic chemistry], Georg Thieme Verlag Stuttgart, fourthedition; volumes 15.1 and 15.2, edited by E. Wünsch.

The affinity tags can also be optionally constructed in the reversesequence, with the Boc protecting group being first of all detached fromderivatives of the formula (IV). The unblocked compounds are thenreacted with isothiocyanates which are correspondingly generated from(VIII) to give compounds of the formula

After the Fmoc protecting group has been detached using piperidine, thecoupling with the derivative of a protein-reactive group or theactivated precursor of the derivative of a protein-reactive groupU-PRG  (V)is carried out in the last step, with compounds of the formula (IX)being obtained in this way as well.

The invention consequently also relates to a process for preparing acompound as claimed in claim 1 in which process

-   -   i) a protected intermediate of the formula (III)

-   -    in which SG and SG′ are two orthogonal protecting groups,    -    is prepared,    -   ii) the protecting group SG is first of all detached from the        intermediate of the formula (III) and, after that, another amino        acid derivative, which carries a protecting group SG, which is        identical to, or different from, the detached protecting group,        on the α-amino function, is attached, with a derivative of the        formula (IV),

-   -    in which SK is the side chain of an amino acid,    -    being obtained,    -   iii) the terminal protecting group SG is detached, with a        conjugate of the formula (VI′)

-   -    being obtained,    -   iv) an affinity ligand A-OH or A-NH₂, or a        hydroxyl-functionalized, carboxyl-functionalized or        amino-functionalized solid phase A-OH or A-NH₂, or an activated        form thereof, is reacted with a compound of the formula (VII)

-   -    in which Y is the optionally branched spacer group    -    which can optionally carry a protecting group, to give the        derivative of the formula (VIII)

-   -   v) the derivative of the formula (VIII) is then converted, after        the prior elimination of an optionally introduced protecting        group, into a corresponding isothiocyanate,    -   vi) the isothiocyanate is then coupled to the conjugate of the        formula (VI′) to give the thiourea of the formula (X′), and

-   -   vii) after the protecting group SG′ has been detached from the        thiourea of the formula (X′), the latter is reacted with the        derivative of a protein-reactive group or the activated        precursor of the derivative of a protein-reactive group of the        formula (V)        U-PRG  (V)    -    in which U is a group which enables PRG to be linked to Z′ or,        where appropriate, to another end group of L,    -    and    -   viii) in an optional last step, protecting groups which may        still be present are eliminated,        with it being possible to carry out the consecutive steps iv)        and v) at any arbitrary time prior to step vi).

The reactions can be carried out under a variety of pressure andtemperature conditions, for example at from 0.5 to 2 bar, and preferablyunder normal pressure, and, respectively, at from −30 to +100° C. andpreferably at from −10 to +80° C., in suitable solvents, such asdimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane,chloroform, lower alcohols, acetonitrile, dioxane or water, or inmixtures of said solvents. As a rule, preference is given to reactionsin DMF, dichloromethane, THF, dioxane/water or THF/dichloromethane, atroom temperature or while cooling with ice and under normal pressure.

Consequently, the described types of affinity tags can be prepared in avariety of ways, both linearly and also by means of coupling blockswhich have already been prefabricated. The modular constructionprinciple makes possible a large number of conceivable combinations andconsequently, when the isotope-labeled modules are selectedappropriately, arbitrary specification of the nature, number andlocation of the isotope labels. Only by using ¹³C and ¹⁵N isotopes inmolecules of the same construction is it possible to obtain an arbitrarynumber, preferably up to 30, isotope labelings. This thereby opens theway to the simultaneous analysis of several, preferably up to 4, complexproteome samples.

EXAMPLES

42 affinity tags of the formula (II), in which A is the acyl residue ofbiotin, and one affinity tag of the formula (II) in which A is an aminogroup-functionalized polymeric support (Example 42), were prepared andare assembled in the following table. In this connection, Z and Z′,insofar as they are present, and with the exception of Z in Example 26and Z′ in Example 43, represent the respective glycine residue such thatit is only the value of k and n, respectively, which are in each caseindicated, with the groups Z¹ and Z² being indicated and defined in thecase of (Z)_(k) in Example 26 and (Z′)_(n) in Example 43, respectively.

S (Z)_(k) [(C₄H₈N₂)L′]_(l) [C₄H₈N₂]_(m) (Z′)_(n) Ex. Y SK of k I L′ m nPRG  1 NHCH₂ Gly 1 0 — 1 1 q = 0  2 NHCH₂ Gly 1 0 — 1 1 q = 1  3 NHCH₂His 1 0 — 1 1 q = 0  4 NHCH₂ Asp 1 0 — 1 1 q = 1  5 NHCH₂ Val 1 0 — 1 1q = 1  6 NHCH₂ Pro 1 0 — 1 1 q = 1  7 NHCH₂ D-Val 1 0 — 1 1 q = 1  8NHCH₂ Gly 0 0 — 1 1 q = 1  9 NHCH₂ Asn 1 0 — 1 1 q = 1 10 NH(CH₂)₂— Gly1 0 — 1 1 q = 1 NHCO 11 NH Gly 1 0 — 1 1 q = 1 12 NHCH₂ D-Val 0 0 — 1 1q = 1 13 NH Gly 1 0 — 1 1 q = 4 14 NH Gly 1 0 — 1 1 q = 2 15 NH Gly 1 0— 1 1 AA 16 NH Gly 1 0 — 1 1 r = 1 17 NH Gly 1 1 L¹ 1 1 q = 1 18 NH Gly1 2 L¹ 1 1 q = 2 19 NH Gly 1 1 CO—CO 1 1 q = 1 20 NH Gly 1 1 L² 1 1 q =1 21 NH His 1 0 — 1 1 q = 2 22 NH Glu 1 0 — 1 1 q = 2 23 NH His 1 0 — 11 q = 2 24 NHCH₂ Gly 0 0 — 1 1 q = 2 25 NHCH₂ His 0 0 — 1 1 q = 2 26NHCH₂ Gly Z¹ 0 — 1 1 q = 2 27 NH Gly 0 1 L¹ 1 1 q = 2 28 NHCH₂ Gly 0 1CO—CO 1 2 q = 2 29 NHCH₂ Gly 0 1 L³ 1 1 q = 2 30 NHCH₂ Gly 0 1 L⁴ 1 1 q= 2 31 NH Gly 0 1 L⁴ 1 1 q = 2 32 NH His 0 1 CO—CO 1 2 q = 2 33 NHCH₂His 0 1 CO—CO 1 2 q = 2 34 NHCH₂ Gly 0 1 CO—CO 1 2 q = 4 35 NH Gly 0 1CO—CO 1 2 q = 4 36 NHCH₂ His 0 1 CO—CO 1 2 q = 4 37 NH His 0 1 CO—CO 1 2q = 4 38 NH Gly 0 1 CO—CO 1 2 q = 2 39 NH Gly 0 1 CO—CO 1 2 q = 2 40 NHGly 0 1 CO—CO 1 2 q = 2 41 NH Gly 0 1 CO—CO 1 2 q = 2 42 COCH₂ Gly 0 1CO—CO 1 2 q = 2 43 NHCH₂ Gly 0 0 — 1 Z² q = 3 44 NHCH₂ Gly 0 0 — 1 Z³ q= 2 45 NHCH₂ Gly 0 0 — 1 Z³ q = 4 46 NH Gly 0 0 — 1 Z³ q = 2 47 NH Gly 00 — 1 Z³ q = 4 48 COCH₂ Gly 1 0 — 1 1 q = 2 L¹ = CO—CH₂—NH—CO; AA =COCHCH₂; Z¹ = NH—(CH₂)₂—CO; L² = CO—CH₂—NH—CO—NH—CH₂—CO; L³ =(CH₂)₃—NH—CO—CO—NH—(CH₂)₃; L⁴ = (CH₂)₂—CO;

Abbreviations employed:

-   Boc tert-butoxycarbonyl-   DIEA diisopropylethylamine (Hünig's base)-   DMAP dimethylaminopyridine-   DMF dimethylformamide-   DMSO dimethyl sulfoxide-   EI electron impact ionization-   ESI electrospray ionization-   Fmoc fluorenyl-9-methoxycarbonyl-   HPLC high performance liquid chromatography-   MALDI matrix-assisted laser desorption ionization-   MS mass spectroscopy-   MTBE methyl tert-butyl ether-   quant. quantitative (i.e. complete) reaction-   RP reverse phase-   RT room temperature-   TCEP tris(carboxyethyl)phosphine-   TFA trifluoroacetic acid-   THF tetrahydrofuran-   TLC thin layer chromatography-   (v/v) indication of concentration in volume per volume-   (w/v) indication of concentration in mass per volume

Unless otherwise expressly indicated, the compositions of solvent andeluent mixtures are in each case given by the names of the components,separated by “/” and, after that, the relative parts by volume. Thus,for example, “acetonitrile/water 10/1” means a mixture of acetonitrileand water in the ratio by volume of 10 to 1.

Eluents which are preferably employed (referred to by indicating ¹⁾etc.):

-   ¹⁾ acetonitrile/water 10/1-   ²⁾ acetonitrile/water 20/1-   ³⁾ dichloromethane/methanol 97.5/2.5-   ⁴⁾ acetonitrile/water/glacial acetic acid 10/1/0.1-   ⁵⁾ acetonitrile/water/glacial acetic acid 5/1/0.2-   ⁶⁾ acetonitrile/water/glacial acetic acid 10/3/1.5-   ⁷⁾ dichloromethane/methanol/aqueous ammonia (17%) 15/2/0.2-   ⁸⁾ acetonitrile/water/glacial acetic acid 10/5/3-   ⁹⁾ dichloromethane/methanol/aqueous ammonia (17%) 15/4/0.5

For the preparation, which is described below, of the exemplary affinitytags, the biotin derivatives of the starting compound series 1 and thepiperazine derivatives of the starting compound series 2 were preparedfirst of all. The piperazine derivatives were then converted into theintermediates of the intermediate series 1 to 3. Finally, theseintermediates were reacted with the biotin derivatives to givecorresponding affinity tags.

Starting Compound Series 1: Biotin Derivatives

1 g (4.09 mmol) of biotin, 500 mg (4.09 mmol) of 4-aminobenzylamine aswell as 830 mg (6.14 mmol) of 1-hydroxy-1H-benzotriazole, 942 mg (4.91mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideand 1587 mg of ethyldiisopropylamine were added together in 40 ml ofDMF. The mixture was stirred overnight at room temperature andconcentrated, and the residue was purified by flash chromatography onsilica gel (elution mixture: dichloromethane/meth-anol/aqueous ammonia(17%) 15/3/0.3). The appropriate fractions were combined and the solventwas evaporated off in vacuo. The residue was stirred up with diethylether and filtered off with suction. 1097 mg (77%) of the intermediatewere obtained [TLC: dichloromethane/methanol/aqueous ammonia (17%)15/4/0.5: R_(f)=0.58] [ESI-MS: m/e=349 (M+H)⁺].

600 mg (1.72 mmol) of this intermediate were dissolved in 40 ml ofdioxane/water 1/1, after which 298 mg (2.58 mmol) of thiophosgene and890 mg of ethyldiisopropylamine were added. The mixture was stirred atroom temperature for 10 min and then concentrated. The target productSC.1.1 was precipitated with diethyl ether fromdichloromethane/methanol.

Yield: 616 mg (92%) [TLC: R_(f)=0.56⁵⁾] [ESI-MS: m/e=391 (M+H)⁺].

Mono-Fmoc-protected p-phenylenediamine was prepared using standardmethods as described, for example, in Houben-Weyl; Methoden derOrganischen Chemie [Methods of organic chemistry]; fourth edition;volume XV parts 1 and 2; Georg Thieme Verlag Stuttgart 1974, or inHans-Dieter Jakubke and Hans Jeschkeit: Aminosäuren, Peptide, Proteine[Amino acids, peptides and proteins]; Verlag Chemie, Weinheim 1982.

200 mg (0.82 mmol) of biotin were taken up in 10 ml of dichloromethaneand 974 mg (8.2 mmol) of thionyl chloride were added. After the mixturehad been stirred for 1 h, it was concentrated and the residue wassubsequently distilled twice with dichloromethane.

The resulting acid chloride (0.81 mmol) was taken up in 30 ml ofdichloromethane, after which 387 mg (4.9 mmol) of pyridine and 242 mg(0.55 mmol) of mono-Fmoc-protected p-phenylenediamine were added. Themixture was stirred at RT for 2 days and the precipitated product wasfiltered off. This resulted in 300 mg (99%) of the intermediate, whichwas used in the next reaction step without any further purification[TLC: R_(f)=0.5¹⁾].

The crude product was taken up in 5 ml of DMF and 500 μl of piperidinewere added. After the mixture had been stirred at room temperature for15 min, it was concentrated and the residue was purified by flashchromatography on silica gel (elution mixture ⁷⁾). The appropriatefractions were combined, the solvent was removed and the residue wasdried in vacuo. 69 mg (39%) of the deprotected intermediate wereobtained.

65 mg (0.19 mmol) of this intermediate were dissolved in 10 ml ofdioxane/water 1/1, after which 33 mg (0.29 mmol) of thiophosgene and 100mg of ethyldiisopropylamine were added. The mixture was stirred at roomtemperature for 10 min and concentrated. The target product SC.1.2 wasprecipitated with diethyl ether from dichloromethane/methanol.

Yield: 65 mg (89%) [TLC: R_(f)=0.43¹⁾] [ESI-MS: m/e 377 (M+H)⁺].

500 mg (3.65 mmol) of 4-aminobenzoic acid were taken up in 20 ml of DMF,after which 872 mg (2.73 mmol) of the hydrochloride of the mono-Fmocprotected ethylenediamine, as well as 739 mg (5.47 mmol) of1-hydroxy-1H-benzotriazole and 839 mg (4.38 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, wereadded. The mixture was stirred at room temperature for 2 h, after whichit was concentrated and the residue was taken up in 200 ml ofdichloromethane. It was then extracted three times by shaking with 200ml of sodium hydrogencarbonate solution. The organic phase wasconcentrated and the residue was purified by flash chromatography onsilica gel (eluent: acetonitrile). The appropriate fractions werecombined and the solvent was evaporated off in vacuo and the residue wasdried. This resulted in 639 mg (59%) of the intermediate [TLC:R_(f)=0.68 ²⁾].

400 mg (1 mmol) of the intermediate were taken up in 10 ml of DMF, afterwhich 500 μl of piperidine were added. After the mixture had beenstirred at room temperature for 15 min, it was concentrated and theresidue was purified by flash chromatography on silica gel (elutionmixture: dichloromethane/methanol/aqueous ammonia (17%) 15/4/0.5). Theappropriate fractions were combined, the solvent was removed and theresidue was dried in vacuo. This resulted in 147 mg (82%) of thedeprotected intermediate [TLC: R_(f)=0.18 ⁵⁾].

191 mg (0.78 mmol) of biotin were taken up in 10 ml of DMF, after which158 mg (1.17 mmol) of 1-hydroxy-1H-benzotriazole and 180 mg (0.94 mmol)of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride wereadded. The mixture was stirred at RT for 10 min and 303 mg ofethyldiisopropylamine and 140 mg (0.78 mmol) of the deprotectedintermediate were added. The mixture was then stirred once again at RTfor 6 h, after which it was concentrated and the crude product wasprecipitated with diethyl ether from dichloromethane. The residue wasseparated off and purified by flash chromatography on silica gel(elution mixture: dichloromethane/methanol/aqueous ammonia (17%)15/3/0.3). The appropriate fractions were combined and the solvent wasevaporated off in vacuo and the residue was dried. This resulted in 222mg (70%) of the intermediate [TLC: dichloromethane/methanol/aqueousammonia (17%) 15/4/0.5: R_(f)=0.47].

200 mg (0.54 mmol) of this intermediate were dissolved in 15 ml ofdioxane/water 1/1, after which 94 mg (0.81 mmol) of thiophosgene and 210mg of ethyldiisopropylamine were added. The mixture was stirred at roomtemperature for 15 min and then concentrated. The target product wasprecipitated with diethyl ether from dichloromethane/methanol.

Yield: 230 mg (95%) [TLC: R_(f)=0.44 ⁵⁾][ESI-MS: m/e=448 (M+H)⁺].

Starting Compound Series 2: Piperazine Derivatives

3.85 g (13 mmol) of Fmoc-glycine, as well as 2.19 g (16.2 mmol) of1-hydroxy-1H-benzotriazole and 2.48 g (13 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride were addedtogether in 80 ml of DMF and the mixture was stirred at RT for 30 min.After that, 2.01 g (10.8 mmol) of Boc-piperazine, dissolved in 40 ml ofDMF, were added dropwise and 2.8 g of ethyldiisopropylamine were alsoadded. The mixture was stirred at RT for 2 h and concentrated. Theresidue was taken up in dichloromethane and shaken twice with water. Theorganic phase was separated off and concentrated and the residue waspurified by flash chromatography on silica gel (eluent: acetonitrile).The appropriate fractions were combined, the solvent was evaporated offin vacuo and the residue was dried. This resulted in 3.91 g (78%) of theintermediate [TLC: R_(f)=0.58 ²⁾].

3.9 g (8.4 mmol) of this intermediate were taken up in 50 ml ofdichloromethane, after which 25 ml of TFA were added. After the mixturehad been stirred at room temperature for 30 min, it was concentrated andthe residue was precipitated with diethyl ether from dichloromethane,filtered off and dried. 4.8 g (93%) of the target product SC.2.1 wereobtained [TLC: R_(f)=0.31 ⁵⁾].

0.53 g (3 mmol) of Boc-glycine, as well as 0.51 g (3.75 mmol) of1-hydroxy-1H-benzotriazole and 0.58 g (3 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride were addedtogether in 40 ml of DMF and the mixture was stirred at RT for 30 min.Subsequently, 1 g of ethyldiisopropylamine was added, after which 1.2 g(2.5 mmol) of the product SC.2.1 were added slowly. The mixture wasstirred overnight at RT and concentrated. The residue was taken up indichloromethane and extracted twice by shaking with sodiumhydrogencarbonate solution. The organic phase was separated off andconcentrated and the residue was purified by flash chromatography onsilica gel (eluent: acetonitrile/water 30/1). The appropriate fractionswere combined, the solvent was evaporated off in vacuo and the residuewas dried. This resulted in 760 mg (58%) of the intermediate [TLC:R_(f)=0.6 ²⁾].

760 mg (1.45 mmol) of this intermediate were taken up in 10 ml ofdichloromethane, after which 5 ml of TFA were added. After the mixturehad been stirred at room temperature for 30 min, it was concentrated andthe residue was precipitated with diethyl ether from dichloromethane,filtered off and dried. 780 mg of the target product SC.2.2 wereobtained (quantitative conversion) [TLC: R_(f)=0.4 ⁵⁾].

3.85 g (13 mmol) of Fmoc-glycine, and also 2.19 g (16.2 mmol) of1-hydroxy-1H-benzotriazole and 2.48 g (13 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, were addedtogether in 80 ml of DMF and the mixture was stirred at RT for 30 min.After that, 2.01 g (10.8 mmol) of Boc-piperazine, dissolved in 40 ml ofDMF, were added dropwise and 2.8 g of ethyldiisopropylamine were alsoadded. The mixture was stirred at RT for 2 h and concentrated. Theresidue was taken off in dichloromethane and extracted twice by shakingwith water. The organic phase was separated off and concentrated and theresidue was purified by flash chromatography on silica gel (eluent:acetonitrile). The appropriate fractions were combined, the solvent wasevaporated off in vacuo and the residue was dried. This resulted in 3.91g (78%) of the intermediate [TLC: R_(f)=0.58 ²⁾].

730 mg (1.57 mmol) of this intermediate were dissolved in 5 ml of DMFafter which 500 μl of piperidine were added. After the mixture had beenstirred at RT for 15 min, it was concentrated and the residue waspurified by flash chromatography on silica gel (eluent ⁷⁾). Theappropriate fractions were combined and the solvent was evaporated offin vacuo. The residue was stirred up with diethyl ether/petroleum ether1/1, after which it was filtered off and dried. This resulted in 222 mg(58%) of the target product SC.2.3 [TLC: R_(f)=0.18 ⁷⁾].

Standard conditions were used to protect the amino group of glycine-¹³C₂with the t-butoxycarbonyl (Boc) protecting group.

Subsequently, 1.175 g (3.83 mmol) of Boc-glycine-¹³C₂ were dissolved indioxane/water 1/1, after which 2.5 g (7.67 mmol) of cesium carbonatewere added. The mixture was lyophilized, after which the product wastaken up in DMF and 2.72 g (19.17 mmol) of iodomethane were then addedto it. After the mixture had been stirred at RT for 2 h, it wasconcentrated and the residue was partitioned between dichloromethane andwater. The organic phase was washed once again with water, after whichit was dried over sodium sulfate and concentrated. 530 mg (72%) of themethyl ester were obtained.

525 mg (2.75 mmol) of the Boc-protected methyl ester were thendeprotected at the amino group with trifluoroacetic acid (yield: 506 mg;90%).

Standard conditions were used to react the resulting product (500 mg)with Boc-glycine-¹³C₂ to give the bilaterally protected dipeptideconjugate containing four ¹³C atoms (yield: 452 mg; 74%).

The Boc group was once again detached from this intermediate using TFA(quantitative conversion).

475 mg (1.8 mmol) of this N-terminally unblocked, ¹³C-labeled dipeptidemethyl ester trifluoroacetate were dissolved in 15 ml of methanol and697 mg (5.4 mmol) of ethyldiisopropylamine were then added. The mixturewas stirred at RT for 3 days, in connection with which thediketopiperazine was formed and precipitated out. It was filtered off,washed with methanol and dried under high vacuum (yield: 138 mg; 65%).

130 mg (1.1 mmol) of the ¹³C-labeled diketopiperazine were taken up in40 ml of THF, under argon, and 284 mg (3.3 mmol) of THF-borane complexwere added. The mixture was stirred under reflux for 12 h and a further284 mg (3.3 mmol) of THF-borane complex were added. After a further 16 hof refluxing, the mixture was allowed to cool down and was quenched with6 ml of 10% hydrochloric acid. The mixture was boiled for a further 30min and then allowed to cool down; it was concentrated and the residuewas subsequently distilled with dichloromethane/methanol. The residuewas then washed with dichloromethane/methanol 4:1 and filtered off. 145mg (81%) of the fourfold ¹³C-labeled piperazine SC.2.4 were obtained[TLC: acetonitrile/water/glacial acetic acid 10/3/1.5: R_(f)=0.23][EI-MS: m/z=90 (M)⁺ radical ion].

This building block was prepared from glycine-¹³C₂—¹⁵N in analogy withSC.2.4.

All the intermediates and end products of the subsequent intermediateseries and examples were prepared from these ¹³C-labeled and ¹³C- and¹⁵N-labeled intermediates SC.2.4 and SC.2.5 in the same way as describedfor the unlabeled ¹²C- and ¹⁴N-analogs.

7.459 g (24.36 mmol) of benzyloxycarbonylglycine-N-hydroxysuccinimideester were added, together with 3 g (34.83 mmol) of piperazine andethyldiisopropylamine, to 70 ml of DMF, and the mixture was stirred atRT for 2 h. The mixture was then concentrated and the residue waspurified by flash chromatography on silica gel (eluent:dichloromethane/methanol/aqueous ammonia (17%) 15/2/0.2). Theappropriate fractions were combined, the solvent was evaporated off invacuo and the residue was dried. 4.04 g (60%) of the target compoundwere obtained [TLC: R_(f)=0.26 ⁵⁾].

The Boc protecting group was introduced into the compound SC.2.5 inaccordance with standard conditions and using 0.5 equivalent of Bocanhydride. Yield: 65% [TLC: R_(f)=0.22⁵⁾].

The Fmoc protecting group was initially introduced into the compoundSC.2.7 in accordance with standard conditions and using Fmoc-Cl, and theBoc protecting group was then detached using trifluoroacetic acid. [TLC:R_(f)=0.32 ⁵⁾].

The protected dipeptide, carrying two ¹⁵N and three ¹³C labels, wasprepared over 5 steps in accordance with standard conditions: first ofall, the Z protecting group was introduced into [bis-¹³C, ¹⁵N]-glycineusing benzyloxycarbonyl chloride in dioxane/1N sodium hydroxide solution(yield: 47%). This amino acid derivative was coupled to[bis-¹³C]-glycine methyl ester (intermediate in the preparation ofSC.2.4) (yield: 77%) and, in the last step, the methyl ester was cleavedusing 2N lithium hydroxide in methanol (yield: 75%). [TLC: R_(f)=0.25⁴⁾] [ESI-MS: m/e=272 (M+H)⁺].

Preparation in analogy with SC.2.6 or from Fmoc-pip.

[TLC: R_(f)=0.35 ⁵⁾].

Intermediate Series 1: Fully Protected Amino Acid-Flanked PiperazineDerivatives

General Formula:

General Directions:

3 mmol of a Boc-protected amino acid derivative, as well as 0.51 g (3.75mmol) of 1-hydroxy-1H-benzotriazole and 0.58 g (3 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, weretogether added to 40 ml of DMF and the mixture was stirred at RT for 30min. 1 g of ethyl diisopropylamine was then added, after which 2.5 mmolof the products SC.2.1 or SC.2.2, dissolved in 40 ml of DMF, were addeddropwise. The mixture was stirred overnight at RT and concentrated. Theresidue was taken up in dichloromethane and this mixture was extractedtwice by shaking with sodium hydrogencarbonate solution. The organicphase was separated off and concentrated and the residue was purifiedeither by precipitation from dichloromethane with diethyl ether or byflash chromatography on silica gel (eluent: acetonitrile/water 30/1).The appropriate fractions were combined, the solvent was evaporated offin vacuo and the residue was dried. The fully protected intermediateswere obtained.

I.1.1 (R=H (Glycine Residue))

-   Starting compounds: Boc-glycine; SC.2.1

Yield: 58% R_(f)=0.45 ¹⁾

I.1.2 (R=D-Valine Residue)

-   Starting compounds: Boc-D-valine; SC.2.1-   Purification: flash chromatography; Eluent: dichloromethane/methanol    98/2

Yield: 76% R_(f)=0.3 ³⁾

Intermediate Series 2: Fully Protected Peptide-Flanked PiperazineDerivatives

General Formula:

The products of the intermediate series 2 were prepared in accordancewith the same general directions as previously described for theintermediate series 1.

I.2.1 (R=H (Glycine (Gly) Residue))

-   Starting compounds: Boc-glycine; SC.2.2

Yield: 86% R_(f)=0.55 ¹⁾

I.2.2 (R=Side Chain Boc-Protected Histidine (His) Residue)

-   Starting compounds: Bis-Boc-histidine-N-hydroxysuccinimide ester;    SC.2.2-   Special features: Instead of the EDCI/HOBT activation, the    hydroxysuccinimide ester was used immediately in this case.    Purification by precipitation.

Yield: 85% R_(f)=0.47 ¹⁾

I.2.3 (R=Side Chain Tert-Butyl Ester-Protected Aspartic Acid (Asp)Residue)

-   Starting compounds: γy-tert-butyl Boc-aspartate; SC.2.2-   Special features: purification by precipitation.

Yield: 68% R_(f)=0.66 ¹⁾

I.2.4 (R=Valine (Val) Residue)

-   Starting compounds: Boc-valine; SC.2.2-   Special features: purification by precipitation.

Yield: 79% R_(f)=0.64 ⁴⁾

I.2.5 (R=Asparagine (Asn) Residue)

-   Starting compounds: Boc-asparagine; SC.2.2-   Special features: purification by flash chromatography using eluent    ²⁾.

Yield: 66% R_(f)=0.47 ¹⁾

I.2.6 (R=Proline (Pro) Residue)

-   Starting compounds: Boc-proline-N-hydroxysuccinimide ester; SC.2.2-   Special features: The activated amino acid derivative was used    instead of EDCI/HOBT. Purification by flash chromatography using    acetonitrile/water 15/1.

Yield: 98% R_(f)=0.55 ⁵⁾

I.2.7 (R=D-Valine (D-Val) Residue)

-   Starting compounds: Boc-D-valine; SC.2.2-   Special features: purification by flash chromatography using    acetonitrile/water 30/1.

Yield: 80% R_(f)=0.64 ¹⁾

I.2.8 (R=β-Alanine Residue)

-   Starting compounds: Boc-p-alanine; SC.2.1-   Special features: SC.2.1 was first of all reacted with Boc-β-analine    and the Boc group was then detached; finally, Boc-glycine was    attached.

Yield: 15% over 3 steps R_(f)=0.45 ⁷⁾

I.2.9 (R=Tert-Butyl Ester-Protected Glutamic Acid Residue)

-   Starting compounds: γ-tert-butyl Boc-glutamate; SC.2.2

Yield: 98% R_(f)=0.75 ¹⁾

Intermediate Series 3: Oligopiperazine Derivatives

1438 mg (7.72 mmol) of Boc-piperazine were dissolved in 100 ml ofdichloromethane, after which 500 mg (3.86 mmol) of oxalyl chloride and625 μl of pyridine were added. After the mixture had been stirred at RTfor 1 h, it was concentrated and the residue was stirred up twice withwater. The remaining solid was sufficiently pure and was dried underhigh vacuum. 1230 mg (75%) were obtained.

Both Boc protective groups were detached from 1230 mg of thisintermediate in accordance with standard conditions (yield: 1330 mg;quantitative conversion).

305 mg (1.31 mmol) of Boc-glycyc-glycine, as well as 242 mg (1.79 mmol)of 1-hydroxy-1H-benzotriazole and 275 mg (1.43 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride weretogether added to 25 ml of DMF and the mixture was stirred at RT for 30min. After that, 543 mg (1.2 mmol) of the bilaterally deprotectedintermediate, dissolved in 5 ml of DMF, were added dropwise and 625 μlof ethyldiisopropylamine were also added. The mixture was stirred at RTfor 1 h and then concentrated. The residue was purified by flashchromatography on silica gel (eluent ⁵⁾). The appropriate fractions werecombined, the solvent was evaporated off in vacuo and the residue wasprecipitated with diethyl ether from dichloromethane. The precipitatewas filtered off and dried under high vacuum. 276 mg (53%) of theintermediate were obtained [TLC: R_(f)=0.32 ⁵⁾].

182 mg (0.61 mmol) of Fmoc-glycine, as well as 124 mg (0.92 mmol) of1-hydroxy-1H-benzotriazole and 141 mg (0.74 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride weretogether added to 20 ml of DMF and the mixture was stirred at RT for 30min. After that, 2270 mg (0.61 mmol) of the above-described intermediatewere added, and 320 μl of ethyldiisopropylamine were also added. Themixture was stirred at RT for 2 h and concentrated. The residue wastaken up in dichloromethane and this solution was extracted twice byshaking with water. The organic phase was separated off, dried oversodium sulfate and concentrated. The residue was then precipitated withdiethyl ether from dichloromethane/methanol 1/1. The precipitate wasfiltered off and dried under high vacuum. This resulted in 195 mg (44%)of the intermediate I.3.1 [TLC: R_(f)=0.52 ⁴⁾].

30 mg (0.123 mmol) of the starting compound SC.2.3 were dissolved indichloromethane, after which 25 mg (0.123 mmol) of 4-nitrophenylchloroformate were added. 172 μl of diisopropylethylamine were addedafter the mixture had been stirred at RT for 30 min, and 59 mg (0.123mmol) of the starting compound SC.2.1 were added after a further 30 min.The mixture was left to stand are RT overnight and then concentrated.The residue was taken up in 20 ml of dichloromethane and this solutionwas extracted by shaking with water. The organic phase was concentratedand the residue was purified by flash chromatography on silica gel(eluent ²⁾). The appropriate fractions were combined, the solvent wasevaporated off in vacuo and the residue was taken up in dioxane/water1/1 and lyophilized. This resulted in 55 mg (70%) of the intermediateI.3.2 [TLC: R_(f)=0.52 ¹⁾] [ESI-MS: m/e=635 (M+H)⁺].

368 mg (0.63 mmol) of the intermediate I.2.1 were taken up in 5 ml ofDMF and 500 μl of piperidine were added. After the mixture had beenstirred at room temperature for 15 min, it was concentrated and theresidue was purified by flash chromatography on silica gel (eluent:dichloromethane/methanol/aqueous ammonia (17%) 15/3/0.3). Theappropriate fractions were combined, the solvent was evaporated off invacuo and the residue was dried. 204 mg (90%) of the target productBoc-Gly-Gly-Pip-Gly were obtained [TLC: dichloromethane/methanol/aqueousammonia (17%) 15/4/0.5: R_(f)=0.28].

70 mg (0.196 mmol) of this intermediate were dissolved in 18 ml ofdichloromethane, after which 59 mg (0.294 mmol) of 4-nitrophenylchloroformate were added. 273 μl of diisopropylethylamine were addedafter the mixture had been stirred at RT for 10 min and 105 mg(0.196.mmol) of the compound SC.2.2 were added after a further 2 h. Themixture was stirred overnight at RT. In connection with this, a solidprecipitated out and was filtered off. 15 mg (10%) of the intermediateI.3.3 were obtained [TLC: R_(f)=0.18 ⁴⁾] [ESI-MS: m/e=806 (M+H)⁺].

The Boc protecting group was first of all detached from the compoundI.3.2 in a known manner. Boc-Gly-Gly-OH was then attached in thepresence of EDCI/HOBT. The target product I.3.4 were obtained in a yieldof 46% over 2 steps. [TLC: R_(f)=0.62 ⁵⁾]

47 mg (0.193 mmol) of the starting compound SC.2.3 were dissolved indichloromethane, after which 39 mg (0.193 mmol) of 4-nitrophenylchloroformate were added. After the mixture had been stirred at RT for10 min, 269 μl of diisopropylethylamine were added and the mixture wasthen stirred at RT for a further 20 min.

In parallel with this, the Boc protecting group was detached from thecompound I.3.2 in a known manner. 125 mg (0.193 mmol) of the deprotectedproduct were then added to the above mixture. The whole was stirred atRT for 4 h and then extracted with 10 ml of water. The organic phase wasconcentrated and the residue was precipitated with diethyl ether fromdichloromethane/methanol 1/1. The precipitate was filtered off withsuction and dried. This resulted in 124 mg (80%) of the intermediate[TLC: R_(f)=0.2 ⁴⁾].

The Boc protecting group was once again detached from this intermediatein a known manner. Boc-Gly-Gly-OH was then attached in the presence ofEDCI/HOBT. The target product was obtained in a yield of 35% over 2steps [TLC: acetonitrile/water/glacial acetic acid 5/1/0.2: R_(f)=0.42][ESI-MS: m/e=918 (M+H)⁺].

Proceeding from I.3.2, the following reactions were carried out inaccordance with standard conditions: Boc elimination usingtrifluoroacetic acid in dichloromethane (yield: 87%), reaction withBoc-glycine-N-carboxylic acid anhydride (yield: 99%) [TLC: R_(f)=0.6¹⁾].

1455 mg (11.46 mmol) of oxalyl chloride were dissolved in 2 ml ofdichloromethane after which 100 mg (0.24 mmol) of Fmoc-piperidine in 10ml of dichloromethane were added. After 1 h, the solvent was distilledoff in vacuo and the residue was subsequently distilled usingdichloromethane.

The residue was then once again taken up in 10 ml of dichloromethane andadded to a solution of 44 mg (0.24 mmol) of Boc-piperidine and 187 mg ofpyridine in 10 ml of dichloromethane. After the mixture had been stirredat RT for 1 h, the solvent was separated off in vacuo and the residuewas purified by flash chromatography on silica gel (eluent:dichloromethane/methanol 97.5/2.5). The appropriate fractions werecombined and the solvent was distilled off in vacuo. 74 mg (57%) of thefully protected intermediate were obtained [TLC: acetonitrile/water20/1: R_(f)=0.6].

The Fmoc protecting group was detached from 72 mg (0.13 mmol) of thisintermediate using piperidine in DMF. In accordance with standardconditions using EDCI/HOBT, the deprotected product was coupled tobenzyloxycarbonylglycylglycine, in the presence ofethyldiisopropylamine, to give the target product. Yield: 82% [TLC:R_(f)=0.5 ⁴⁾].

713 mg (2.93 mmol) of N-3-aminopropyl-N′-tert-butoxycarbonyl piperazineand 2.3 g of ethyldiisopropylamine were initially introduced in 100 mlof dichloromethane, after which 225 mg (1.78 mmol) of oxalyl chloridewere added dropwise. After the mixture had been stirred at RT for 1 h,it was diluted with a further 100 ml of oxalyl chloride and thenextracted three times by shaking with 5% strength sodiumhydrogencarbonate solution. The organic phase was dried over sodiumsulfate and concentrated. The residue was digested with diethyl etherand the product was filtered off. The mother liquor was precipitatedonce again with petroleum ether. 515 mg (54%) of the fully protectedintermediate were obtained [TLC: R_(f)=0.3 ⁶⁾].

The Boc protecting group was detached from the above usingtrifluoroacetic acid (quantitative reaction). In accordance withstandard conditions using EDCI/HOBT, the deprotected product was thencoupled to Boc-glycine, in the presence of ethyldiisopropylamine, togive the target product. Yield: 42% [TLC: R_(f)=0.61 ⁹⁾] [ESI-MS:m/e=655 (M+H)⁺].

793 mg (1.38 mmol) of the compound from Example I.3.7 were dissolved in40 ml of methanol and 15 ml of THF and hydrogenated overpalladium/active charcoal (10% Pd). After 1 h, the catalyst wasseparated off and the solvent was evaporated. The residue was taken upin dioxane/water and lyophilized. This resulted in 512 mg (84%) of thetarget compound [TLC: R_(f)=0.17 ⁵⁾].

49.9 mg (0.272 mmol) of ω-maleimidobutyric acid, as well as 46 mg (0.34mmol) of 1-hydroxy-1H-benzotriazole and 52 mg (0.272 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, weretogether added to 20 ml of DMF and the mixture was stirred at RT for 90min. After that, 100 mg (0.227 mmol) of the compound from Example 1.3.9were added, and 120 μl of ethyldiisopropylamine were also added. Themixture was stirred at RT for 6 h and concentrated. The residue wastaken up in dichloromethane and this solution was extracted three timesby shaking with water. The organic phase was separated off, dried oversodium sulfate and concentrated. The residue was then precipitated withdiethylether from dichloromethane. The precipitate was filtered off anddried under high vacuum. 78 mg (yield: 57%) of the intermediate wereobtained.

The Boc protecting group was detached from the above usingtrifluoroacetic acid in order to obtain the target compound(quantitative reaction) [TLC: R_(f)=0.28 ⁶⁾].

The preparation was effected in analogy with Example I.3.10, proceedingfrom I.3.9.

Yield: 72% [TLC: R_(f)=0.38 ⁶⁾].

500 mg (1.94 mmol) of N-2-carboxyethyl-N′-tert-butoxycarbonylpiperazinewere coupled to 537 mg (1.94 mmol) of benzyloxycarbonylglycylpiperazine(compound SC.2.6) in accordance with standard conditions usingEDCI/HOBT. 630 mg (yield: 63%) of the fully protected intermediate wereobtained [TLC: R_(f)=0.4 ⁵⁾].

400 mg of the intermediate were hydrogenated in 40 ml of methanol andover palladium/active charcoal (10% Pd). After 3 h, the catalyst wasseparated off and the solvent was evaporated and the residue dried. 401mg (yield: 95%) were obtained [TLC: R_(f)=0.2 ⁶⁾].

This intermediate was coupled to ω-maleimidobutyric acid as described inExample I.3.10 and using EDCI/HOBT. The Boc group was then detachedusing trifluoroacetic acid. The target compound was obtained in a yieldof 46% over 2 steps [TLC: R_(f)=0.21 ⁸⁾].

The preparation was effected in analogy to the example I.3.7 using thebuilding blocks:

-   Fmoc-piperidine-   Oxalyl chloride-   Boc-piperidine-   Compound from Example SC.2.9.

Yield: 52% over 4 steps [TLC: R_(f)=0.4 ⁷⁾][ESI-MS: m/e=580 (M+H)⁺].

The preparation was effected in analogy with the Example I.3.7 using thebuilding blocks:

-   Fmoc-piperidine-   Oxalyl chloride-   Compound from Example SC.2.7-   Compound from Example SC.2.9.

Yield: 38% over 4 steps [TLC: R_(f)=0.4 ⁷⁾][ESI-MS: m/e=586 (M+H)⁺].

The preparation was effected in analogy with Example I.3.7 using thebuilding blocks:

-   Compound from Example SC.2.8-   Oxalyl chloride-   Compound from Example SC.2.7-   Compound from Example SC.2.9.

Yield: 41% over 4 steps [TLC: R_(f)=0.55 ⁴⁾]. [ESI-MS: m/e=592 (M+H)⁺].

The preparation was effected in analogy with Examples I.3.9 and I.3.10,proceeding from:

-   compound from Example I.3.13

Yield: 60% over 3 steps [TLC: R_(f)=0.16 ⁵⁾]

The preparation was effected in analogy with Examples I.3.9 and I.3.10,proceeding from:

-   compound from Example I.3.14

Yield: 66% over 3 steps [TLC: R_(f)=0.16 ⁵⁾].

The preparation was effected in analogy with Examples I.3.9 and I.3.10,proceeding from:

-   compound from Example I.3.15

Yield: 49% over 3 steps [TLC: R_(f)=0.16 ⁵⁾]

EXAMPLES OF ACID-CLEAVABLE AFFINITY TAGS Example 1 Preparation Process(Variant A)

368 mg (0.63 mmol) of the intermediate I.2.1 were taken up in 5 ml ofDMF, after which 500 μl of piperidine were added. After the mixture hadbeen stirred at room temperature for 15 min, it was concentrated and theresidue was purified by flash chromatography on silica gel (eluent:dichloromethane/methanol/aqueous ammonia (17%) 15/3/0.3). Theappropriate fractions were combined, the solvent was evaporated in vacuoand the residue was dried. 204 mg (90%) of the target product wereobtained [TLC: dichloromethane/methanol/17% ammonia 15/4/0.5R_(f)=0.28].

26 mg (73 μmol) of this intermediate were taken up in 10 ml of DMF,after which 18.4 mg (73 μmol) of maleimidoaceticacid-N-hydroxysuccinimide ester and 19 mg of ethyldiisopropylamine wereadded and the mixture was stirred at RT for 1 h. Alternatively, it isalso possible to couple maleimidoacetic acid to the amine component inthe presence of EDCI/HOBT. The solvent was evaporated and the residuewas purified by flash chromatography on silica gel (eluent ²⁾). Theappropriate fractions were combined, the solvent was evaporated in vacuoand the residue was dried. 34 mg (95%) of the desired product wereobtained [TLC: R_(f)=0.17 ²⁾].

33 mg (67 μmol) of this intermediate were taken up in 5 ml ofdichloromethane, after which 1 ml of TFA was added. After the mixturehad been stirred at room temperature for 15 min, it was concentrated andthe residue was precipitated with diethyl ether from dichloromethane.After filtration and drying, 33 mg (97%) of the desired product wereobtained as the trifluoroacetic acid salt [TLC:acetonitrile/water/glacial acetic acid 10/3/1.5 R_(f)=0.22] [ESI-MS:m/e=395 (M+H)⁺].

32 mg (63 μmol) of the deprotected intermediate and 26 mg (70 μmol) ofthe isothiocyanate SC.1.1 from the starting compound series 1 weredissolved in 5 ml of DMF after which 37 μl of ethyldiisopropylamine wereadded and the mixture was then stirred at RT for 4 h. The mixture wasconcentrated and the residue was stirred up with water and filtered offwith suction. The residue was separated off and then treated three timeswith dichloromethane/methanol 1:1, and a further twice with methanol, inan ultrasonic bath. After drying, 19 mg (35%) of the target compoundwere obtained [TLC: R_(f)=0.5 ⁵⁾] [ESI-MS: m/e=785 (M+H)⁺].

Example 2 Preparation Process (Variant B)

182 mg (0.31 mmol) of the compound I.1.1 were taken up in 15 ml ofdichloromethane after which 1 ml of TFA was added. After the mixture hadbeen stirred at room temperature for 15 min, it was concentrated and theresidue was precipitated with diethyl ether from dichloromethane. Afterfiltration and drying, 175 mg (94%) of the desired product were obtainedas the trifluoroacetic acid salt [TLC: R_(f)=0.2 ⁵⁾].

170 mg (286 μmol) of the deprotected intermediate were initiallyintroduced in 10 ml of DMF, after which 112 mg (286 μmol) of theisothiocyanate SC.1.1 from the starting compound series 1 and 150 μl ofethyldiisopropylamine were added and the mixture was then stirredovernight at RT. The mixture was concentrated and the residue wasstirred up with 10 ml of water and filtered off with suction. Theresidue was separated off and then stirred up with dichloromethane.After filtration and drying, 244 mg (98%) of the desired compound wereobtained [TLC: R_(f)=0.23 ⁴⁾].

240 mg (0.276 mmol) of this intermediate were taken up in 10 ml of DMFafter which 500 μl of piperazine were added. After the mixture had beenstirred at room temperature for 30 min, it was concentrated and theresidue was digested with dichloromethane. It was then filtered off andthe filter residue was suspended in a mixture of 5 ml of DMF and 10 mlof dichloromethane. After 10 ml of diethyl ether had been added, theproduct precipitated out completely and was filtered off and dried. 135mg (76%) of the target product were obtained [TLC:acetonitrile/water/glacial acetic acid 10/3/1.5 R_(f)=0.2].

30 mg (46 μmol) of this intermediate and 18 mg of ethyldiisopropylaminewere added to a solution of 8 mg (46 μmol) of maleimidopropionic acid,9.4 mg (70 μmol) of HOBT and 11 mg (56 μmol) of EDCI in 10 ml of DMF,which solution had previously reacted for 30 min, and the whole was thenstirred overnight at RT. The solvent was evaporated off and the residuewas stirred up with 5 ml of water. The solid residue was then stirred upwith 5 ml of dichloromethane/methanol 1:1, after which 5 ml of diethylether were added. The precipitated product was dried under high vacuum.28 mg (76%) were obtained [TLC: R_(f)=0.4 ⁵⁾] [ESI-MS: m/e=799 (M+H)⁺].

The following examples were prepared in an analogous manner to Example 1(Variant A) or Example 2 (Variant B). The variant is indicated below ineach case.

Example 3

-   Starting compounds: I.2.2, SC. 1.1; Variant A

Yield: 33% over 4 steps, then conversion into the hydrochloride using1.5 equivalents of a 0.1 M aqueous solution of hydrochloric acidR_(f)=0.3 ⁶⁾ [ESI-MS: m/e=865 (M+H)⁺]

Example 4

-   Starting compounds: I.2.3, SC.1.1; Variant A-   Special features: EDCI/HOBT was used to attach maleimidopropionic    acid instead of maleimidoacetic acid N-hydroxysuccinimide ester

Yield: 33% over 4 steps R_(f)=0.33 ⁵⁾ [ESI-MS: m/e=857 (M+H)⁺]

Example 5

-   Starting compounds: I.2.4, SC.1.1; Variant A-   Special features: EDCI/HOBT was used to attach maleimidopropionic    acid instead of maleimidoacetic acid N-hydroxysuccinimide ester

Yield: 30% over 4 steps R_(f)=0.55 ⁵⁾ [ESI-MS: m/e=841 (M+H)⁺]

Example 6

-   Starting compounds: I.2.6, SC.1.1; Variant A-   Special features: EDCI/HOBT was used to attach maleimidopropionic    acid instead of maleimidoacetic acid N-hydroxysuccinimide ester

Yield: 23% over 4 steps R_(f)=0.44 ⁵⁾ [MALDI-MS: m/e=861 (M+Na)⁺]

Example 7

-   Starting compounds: I.2.7, SC.1.1; Variant A-   Special features: EDCI/HOBT was used to attach maleimidopropionic    acid instead of maleimidoacetic acid N-hydroxysuccinimide ester

Yield: 37% over 4 steps R_(f)=0.56 ⁵⁾ [ESI-MS: m/e=841 (M+H)⁺]

Example 8

-   Starting compounds: I.1.1, SC.1.1; Variant A

Yield: 70% over 4 steps R_(f)=0.53 ⁵⁾ [ESI-MS: m/e=742 (M+H)⁺]

Example 9

-   Starting compounds: I.2.5, SC.1.1; Variant B

Yield: 57% over 4 steps R_(f)=0.28 ⁵⁾ [MALDI-MS: m/e 878 (M+Na)⁺]

Example 10

-   Starting compounds: I.2.1, SC.1.3; Variant A

Yield: 34% over 4 steps R_(f)=0.25 ⁵⁾ [MALDI-MS: m/e=878 (M+Na)⁺]

Example 11

-   Starting compounds: I.2.1, SC.1.2; Variant A

Yield: 31% over 4 steps R_(f)=0.4 ⁵⁾ [MALDI-MS: m/e 807 (M+Na)⁺]

Example 12

-   Starting compounds: I.1.2, SC.1.1; Variant A

Yield: 34% over 4 steps R_(f)=0.38 ⁴⁾ [ESI-MS: m/e=784 (M+H)⁺]

Example 13

-   Starting compounds: I.2.1, SC.1.2; Variant B-   Special features: Instead of attaching maleimidopropionic acid in    the presence of EDCI/HOBT, the amine precursor of the end product    was, in this present case, reacted, in the last step, with    ε-maleimidocaprylic acid in the presence of EDCI/HOBT.

Yield: 15 mg (19% over 4 steps) R_(f)=0.5 ⁵⁾ [ESI-MS: m/e=827 (M+H)⁺]

Example 14

-   Starting compounds: I.2.1, SC.1.2; Variant B-   Special features: Instead of attaching maleimidopropionic acid in    the presence of EDCI/HOBT, the amine precursor of the end product    was, in this present case, reacted, in the last step, with    ε-maleimidobutyric acid in the presence of EDCI/HOBT.

Yield: 12% over 4 steps R_(f)=0.5 ⁵⁾ [ESI-MS: m/e=799 (M+H)⁺]

Example 15

-   Starting compounds: I.2.1, SC.1.2; Variant B-   Special features: Instead of attaching maleimidopropionic acid in    the presence of EDCI/HOBT, the amine precursor of the end product    was, in this present case, reacted, in the last step, with acryloyl    chloride (6 equiv.) in dichloromethane in the presence of 2    equivalents of pyridine. The resulting target compound was purified    by flash chromatography (eluent: acetonitrile/water 10/1).

Yield: 3% over 4 steps R_(f)=0.15 ⁴⁾ [ESI-MS: m/e=688 (M+H)⁺]

Example 16

-   Starting compounds: I.2.1, SC.1.2; Variant B-   Special features: Instead of attaching maleimidopropionic acid in    the presence of EDCI/HOBT, the amine precursor of the end product    was, in this present case, reacted, in the last step, with    chloroacetyl chloride in dichloromethane in the presence of 2    equivalents of pyridine.

Yield: 22% over 4 steps R_(f)=0.12 ¹⁾ [MALDI-MS: m/e=732 (M+Na)⁺]

Example 17

-   Starting compounds: I.3.4, SC.1.2; Variant B

Yield: 58% over 4 steps R_(f)=0.2 ⁵⁾ [ESI-MS: m/e=954 (M+H)⁺]

Example 18

-   Starting compounds: I.3.5, SC.1.2; Variant B

Example 19

-   Starting compounds: I.3.1, SC.1.2; Variant B

Yield: 83% over 4 steps R_(f)=0.4⁵⁾ [ESI-MS: m/e=925 (M+H)⁺]

Example 20

-   Starting compounds: I.3.3, SC.1.2; Variant B

Example 21

-   Starting compounds: I.2.2, SC.1.2; Variant B

Yield: 56% over 4 steps R_(f)=0.3 ⁸⁾ [ESI-MS: m/e=879 (M+H)⁺]

Example 22

-   Starting compounds: I.2.9, SC.1.2; Variant A

Yield: 26% over 4 steps R_(f)=0.5 ⁵⁾ [ESI-MS: m/e=871 (M+H)⁺]

Example 23

The preparation was effected by converting the compound from Example 21into the hydrochloride using 0.1 M aqueous HCl R_(f)=0.23 ⁶⁾ [ESI-MS:m/e=879 (M+H)⁺]

Example 24

-   Starting compounds: I.1.1, SC.1.1; Variant B

Yield: 47% over 4 steps R_(f)=0.5 ⁵⁾ [ESI-MS: m/e 756 (M+H)⁺]

Example 25

-   Starting compound: SC.2.3, after which there then came the following    standard reactions:

Linkage to ω-maleimidobutyric acid in the presence of EDCI/HOBT (61%),Boc elimination (88%),

Linkage to bis-Boc-histidine N-hydroxysuccinimide ester (52%), Bocelimination (69%),

Reaction with SC.1.1 (96%).

R_(f)=0.4 ⁶⁾ [ESI-MS: m/e=836 (M+H)⁺]

Example 26

-   Starting compounds: I.2.8, SC.1.1; Variant B

Yield: 23% over 4 steps R_(f)=0.3 ⁵⁾ [ESI-MS: m/e=827 (M+H)⁺]

Example 27

-   Starting compounds: I.3.6, SC.1.2; Variant B

Yield: 27% over 4 steps R_(f)=0.26 ⁵⁾ [ESI-MS: m/e=911 (M+H)⁺]

Example 28

-   Starting compound: I.3.10, after which there then came the following    standard reactions:

Linkage to Boc-glycine N-carboxylic acid anhydride (71%),

Boc elimination using trifluoroacetic acid (80%)

Linkage to SC.1.1 (54%).

R_(f)=0.38 ⁵⁾ [ESI-MS: m/e=953 (M+H)⁺]

Example 29

-   Starting compound: I.3.8, after which there then came the following    standard reactions:

Boc elimination at both terminal amino groups using trifluoroacetic acid(quant.), reaction with ½ equiv. of the compound SC.1.1 to give themonothiourea (28%) [R_(f)=0.15 ⁹⁾]

Reaction with ω-maleidobutyric acid in the presence of EDCI/HOBT (50%)[ESI-MS: m/e=1010 (M+H)⁺]

Example 30

-   Starting compound: I.3.12, after which there then came the following    standard reactions:

Reaction with Boc-glycine N-carboxylic acid anhydride (84%),

Boc elimination using trifluoroacetic acid (quant.), [R_(f)=0.28 ⁸⁾]

Reaction with SC.1.1 (60%), [R_(f)=0.46 ⁶⁾][ESI-MS: m/e=896 (M+H)⁺]

Example 31

The preparation was effected in analogy with the compound from Example30.

Reaction with SC.1.2 instead of SC.1.1 (52%), [R_(f)=0.55 ⁶⁾][ESI-MS:m/e=882, (M+H)⁺]

Example 32

-   Starting compound: I.3.10, after which there then came the following    standard reactions:

Reaction with bis-Boc-histidine H-hydroxysuccinimide ester (39%)

Boc elimination using trifluoroacetic acid (87%), [R_(f)=0.44⁸⁾]

Reaction with SC.1.2 (45%), [R_(f)=0.44 ⁶⁾][ESI-MS: m/e=1019 (M+H)⁺]

Example 33

The preparation was effected in analogy with the compound from Example32.

Reaction with SC.1.1 instead of SC.1.2 (35%), [R_(f)=0.3 ⁶⁾][ESI-MS:m/e=1033 (M+H)⁺]

Example 34

-   Starting compound: I.3.11, after which there then came the following    standard reactions:

Reaction with Boc-glycine N-carboxylic acid anhydride (69%)

Boc elimination using trifluoroacetic acid (quant.), [R_(f)=0.3 ⁶⁾]

Reaction with SC.1.1 (97%), [R_(f)=0.44 ⁶⁾][ESI-MS: m/e=981 (M+H)⁺]

Example 35

The preparation was effected in analogy with the compound from Example34

Reaction with SC.1.2 instead of SC.1.1 (73%), [R_(f)=0.48 ⁵⁾][ESI-MS:m/e=967 (M+H)⁺]

Example 36

-   Starting compound: I.3.11, after which there then came the following    standard reactions:

Reaction with bis-Boc-histidine N-hydroxysuccinimide ester (41%)

Boc elimination using trifluoroacetic acid (quant.), [R_(f)=0.12 ⁶⁾]

Reaction with SC.1.1 (90%), [R_(f)=0.38 ⁶⁾][ESI-MS: m/e 1061 (M+H)⁺]

Example 37

The preparation was effected in analogy with the compound from Example36.

Reaction with SC.1.2 instead of SC.1.1 (73%), [R_(f)=0.4 ⁶⁾][ESI-MS:m/e=1047 (M+H)⁺]

Example 38

The preparation was effected in analogy with the compound from Example28.

Reaction with SC.1.2 instead of SC.1.1 (72%), [R_(f)=0.45 ⁵⁾][ESI-MS:m/e=(M+H)⁺]

Together with the affinity tags from Examples 39, 40 and 41, thisaffinity tag forms an affinity tag quadruplet which makes it possible toanalyze four proteome samples in parallel.

Example 39

-   Starting compound: I.3.16, after which there then came the following    standard reactions:

Linkage to Boc-glycine N-carboxylic acid anhydride (81%),

Boc elimination using trifluoroacetic acid (98%),

Linkage to SC.1.2 (81%)

R_(f)=0.5 ⁵⁾ [ESI-MS: m/e=944 (M+H)⁺]

Example 40

-   Starting compound: I.3.17, after which there then came the following    standard reactions:

Linkage to Boc-glycine N-carboxylic acid anhydride (80%),

Boc elimination using trifluoroacetic acid (quant.),

Linkage to SC.1.2 (74%)

R_(f)=0.5 ⁵⁾ [ESI-MS: m/e=950 (M+H)⁺]

Example 41

-   Starting compound: I.3.18, after which there then came the following    standard reactions:

Linkage to Boc-glycine N-carboxylic acid anhydride (94%),

Boc elimination using trifluoroacetic acid (98%),

Linkage to SC.1.2 (38%)

R_(f)=0.5 ⁵⁾ [ESI-MS: m/e=956 (M+H)⁺]

Example 42

-   Starting compound: I.3.10, after which there then came the following    standard reactions:

Linkage to Boc-glycine N-carboxylic acid anhydride (78%),

Boc elimination using trifluoroacetic acid (93%) [R_(f)=0.2 ⁶⁾]

In parallel with this, 100 mg (0.026 mmol) of NovaSyn TG resin01-64-0043 were washed three times with DMF. After that, 28 mg (0.08mmol) of 4-(Fmoc-amino)benzoic acid, 30 mg of HATU and 20 mg ofdiisopropylethylamine in 2 ml of DMF were added and the mixture wasstirred overnight at RT. The resin was then washed four times with DMF.After that, standard conditions were used to detach the Fmoc group andthe resin was washed four times with DMF. 2 ml of dioxane/water 1/1 and10 μl of thiophosgene were added. After 1 h, 200 μl ofethyldiisopropylamine were added and, after a further 1 h, the resin waswashed with water, dioxane and DMF. 35 mg of the previously preparedamine component were added in 2 ml of DMF and 25 μl ofethyldiisopropylamine. After 2 h, the resin was washed in each casetwice with DMF and THF and dried.

Example 43

-   Starting compound: SC.2.10, after which there then came the    following standard reactions:

Reaction with Z-Glu(tBu)-Osu (33%),

Hydrogenation over Pd—C (92%),

Reaction with Z-Leu in the presence of EDCI/HOBT (88%),

Hydrogenation over Pd—C (85%),

Linkage to ω-maleimidobutyric acid in the presence of EDCI/HOBT (66%),

Boc elimination (88%),

Reaction with SC.1.1 (88%).

R_(f)=0.26 ⁴⁾ [ESI-MS: m/e=941 (M+H)⁺]

Example 44

-   Starting compound: SC.2.10, after which there then came the    following standard reactions:

Reaction with Fmoc-Leu-Leu in the presence of EDCI/HOBT (58%),

Boc elimination (78%),

Reaction with SC.1.1 (90%),

Fmoc elimination using piperidine in DMF (68%),

Linkage to ω-maleimidobutyric acid in the presence of EDCI/HOBT (72%),

R_(f)=0.68 ⁵⁾ [FAB-MS: m/e=925 (M+H)⁺]

Example 45

Preparation in analogy with Example 44; however, in the last step, thelinkage takes place to ω-maleimidocaprylic acid in the presence ofEDCI/HOBT (61%),

R_(f)=0.4 ⁴⁾ [FAB-MS: m/e=953 (M+H)⁺]

Example 46

-   Starting compound: SC.2.10, after which there then came the    following standard reactions:

Reaction with Fmoc-Leu-Leu in the presence of EDCI/HOBT (58%),

Boc elimination (78%),

Reaction with SC.1.2 (72%),

Fmoc elimination using piperidine in DMF (96%),

Linkage to ω-maleimidobutyric acid in the presence of EDCI/HOBT (92%),

R_(f)=0.4 ⁴⁾ [ESI-MS: m/e=911 (M+H)⁺]

Example 47

Preparation in analogy with Example 46; however, in the last step, thelinkage takes place to ω-maleimidocaprylic acid in the presence ofEDCI/HOBT (61%),

R_(f)=0.5 ⁴⁾ [FAB-MS: m/e=939 (M+H)⁺]

Example 48

-   Starting compound 1.2.1, after which there then came the following    standard reactions:

Boc elimination using trifluoroacetic acid (94%) [R_(f)=0.2 ⁵⁾]. Thisthereby gives the amine component A.

In parallel with this, 42 mg (0.04 mmol) of aminopropyl silica gel(Aldrich, 36425-8, loading 0.95 mmol/g) were suspended in 2 ml of DMF,after which 45 mg (0.12mmol) of 4-(Fmoc-amino)phenylacetic acid, 2 μl ofdiisopropylethylamine, 15 mg (0.12 mmol) of diisopropylcarbodiimide and16 mg (0.12 mmol) of HOBT were added consecutively. The mixture isallowed to stand at RT overnight, after which the resin is washed fourtimes with DMF.

The Fmoc group is then detached using 2 ml of 20% piperidine in DMF andthe resin is washed in each case four times with DMF and dioxane.

1 ml of dioxane and 45 μl of thiophosgene are then added. After 1 h, 900μl of ethyl diisopropylamine are added and, after a further 1 h, theresin is washed in each case three times with dioxane, DMF and DCM.

47 mg (0.08 mmol) of the previously prepared amine component are addedin 2 ml of DMF and 40 μl of ethyldiisopropylamine. The mixture is leftto stand overnight at RT and the resin is then washed four times withDMF.

The Fmoc group is detached once again using 2 ml of 20% piperidine inDMF and the resin is washed four times with DMF.

1 ml of DMF is added, after which 22 mg (0.12 mmol) of4-maleimidobutyric acid, 15 mg (0.12 mmol) of diisopropylcarbodiimideand 16 mg (0.12 mmol) of HOBT are added consecutively and the mixture isstirred overnight at RT. The resin is then washed in each case threetimes with DMF, DCM and THF.

Investigations Involving Protein Analysis

Coupling the Affinity Tags to SDS-7 and Description of the OperationalProcedure for the Affinity Tag from Example 11

A mixture of seven proteins, which are also used as a size standard ingel electrophoresis (SDS-7 markers, Sigma-Aldrich GmbH, Taufkirchen) wasused as the sample.

24 μg of the protein mixture were dissolved in 5 μl of buffer 1 and thissolution was diluted with 135 μl of buffer 3. The proteins weredenatured by heating at 100° C. for 3 minutes. In order to reduce thecysteines which were present, 3 μl of reducing solution were added andthe mixture was incubated at 100° C. for 10 minutes. In order to reactthe free cysteines with the affinity tag from Example 11, 5 μl ofderivatizing solution were then added and the mixture was incubated at37° C. for 90 minutes.

After the derivatization, 3 μl of trypsin solution were added. Theproteins are cleaved overnight (approx. 17 hours) at 37° C.

Buffer 1: 50 mM Tris-HCl, pH 8.3; 5 mM EDTA; 0.5% (w/v) SDS

Buffer 2: 10 mM NH₄acetate, pH 7

Buffer 3: 50 mM Tris-HCl, pH 8.3; 5 mM EDTA

Reducing solution: 50 mM TECP in buffer 2

Derivatizing solution: 30 μg of affinity tag (Example 11)/μl in DMSO

Trypsin solution: 1 mg of trypsin (Promega GmbH, Mannheim)/ml in buffer3

Affinity Purification of Derivatized Peptides

The affinity columns (Monomeric Avidin, Perbio Science Deutschland GmbH,Bonn), having a column volume of 200 μl, were prepared freshly prior tothe purification and made ready by means of the following washing steps:

-   -   two column volumes of 2×PBS    -   four column volumes of 30% (v/v) acetonitrile/0.4% (v/v)        trifluoroacetic acid    -   seven column volumes of 2×PBS    -   four column volumes of 2 mM biotin in 2×PBS    -   six column volumes of 100 mM glycine, pH 2.8    -   six column volumes of 2×PBS

Prior to loading, 30 μl of sample were diluted with 30 μl of 2×PBS,after which the diluted sample was loaded onto the column. After that,the following washing steps were carried out in order to remove theunbiotinylated peptides:

-   -   six column volumes of 2×PBS    -   six column volumes of PBS    -   six column volumes of 50 mM ammonium hydrogencarbonate/20% (v/v)        methanol    -   one column volume of 0.3% (v/v) formic acid

The sample was eluted by means of the following steps:

-   -   three column volumes of 0.3% (v/v) formic acid    -   three column volumes of 30% (v/v) acetonitrile/0.4% (v/v)        trifluoroacetic acid

The eluate was evaporated down to dryness and only dissolved once againshortly before carrying out the mass spectrometric analysis.

PBS: 10× stock solution, GibcoBRL, Cat. No. 14200-067

Mass-Spectrometric Analysis

An ion trap mass spectrometer (LCQdeka, ThermoFinnigan, San Jose) whichwas connected directly to a high pressure liquid chromatographyappliance (LC-MS) was used for analyzing the peptides. A reversed-phasecolumn (C₁₈ phase) was used as the separation column. The peptides weredissolved in eluent A (0.025% (v/v) trifluoroacetic acid) and injected.They were eluted with a gradient of eluent B (0.025% (v/v)trifluoroacetic acid/84% (v/v) acetonitrile). The eluting peptides wererecognized automatically by the acquisition software in the instrumentand fragmented for identification. In this way, it was possible todetermine the identities of the peptides unambiguously.

FIG. 1 shows an example of a fragment spectrum of a peptide from thisanalysis. The observed pattern identifies the peptide unambiguously asbeing the peptide having the sequence FLDDDLTDDIMCVK from lactalbumin,which was a constituent of the sample. The mass of the peptide, and itsfragmentation, confirm that the affinity tag was cleaved by acid in theexpected manner.

In all, 19 different peptides from the sample, all of which peptidescarried the expected mass of the affinity tag residue in the samemanner, were identified in one analysis. No cysteine-containing peptidewhich was still carrying a complete affinity tag was identified.

FIG. 1: Fragment spectrum of a peptide which was derivatized with thecompound from Example 11 after the peptide had been isolated usingavidin, i.e. possessing an acid-cleaved affinity tag.

Coupling the Affinity Tags to Proteins and Description of theOperational Procedure for the Affinity Tags in Examples 38 to 41

A mixture of seven proteins, which are also used as size standards ingel electrophoresis (SDS-7 markers, Sigma-Aldrich GmbH, Taufkirchen),was used as the sample. The two samples to be compared containedidentical quantities of the following proteins:

In addition, human interleukin-4, which was not present in sample 2, wasadded to sample 1.

About 240 μg of protein, which were distributed approximately equallybetween the abovementioned 7 or, respectively, 8 proteins, were used ineach sample. The SDS-7 samples were first of all dissolved in 10 ml ofbuffer 1 and diluted with 95 μl of buffer 3. 10 μl of interleukin-4solution were then also added to sample 1. The samples were made up to200 μl with buffer 3 and in each case divided in half (samples 1a and 1band 2a and 2b, respectively).

The proteins were then denatured by heating them at 100° C. for 3minutes. In order to reduce the cysteines which were present, 3 μl ofreducing solution were added and the mixtures were incubated at 100° C.for 10 minutes. In order to react the three cysteines with the affinitytags from the examples, 5 μl of derivatizing solution were added to eachsample and the mixtures were incubated at 37° C. for 120 minutes. Allthe 4 samples were then mixed and half of the total sample was subjectedto further processing.

Sample 1a: reaction with Example 38

Sample 1b: reaction with Example 41

Sample 2a: reaction with Example 39

Sample 2b: reaction with Example 40

After the reaction had taken place, the proteins were precipitated, at−20° C. for 15 minutes, by adding four times the volume of ice-coldacetone/ethanol 1:1. The sediment was washed once withacetone/ethanol/water 4:4:2 and then dried in vacuo. The sample wasdissolved in 10 μl of buffer 1 and diluted with 260 μl of buffer 3. Inorder to cleave the proteins, 40 μl of trypsin solution were added andthe sample was incubated at 37° C. for 1 h. After that, the sample washeated briefly at 95° C. in order to inactivate the enzyme.

Buffer 1: 50 mM Tris-HCl, pH 8.3; 5 mM EDTA; 0.5% (w/v) SDS

Buffer 2: 10 mM NH₄acetate, pH 7

Buffer 3: 50 mM Tris-HCl, pH 8.3; 5 mM EDTA

Reducing solution: 50 mM TCEP in buffer 2

Derivatizing solution: 36 μg of affinity tag (Examples 38–41)/μl in DMSO

Trypsin solution: 1 mg of trypsin (Promega GmbH, Mannheim)/ml in buffer3

Affinity Purification of Derivatized Peptides

In order to selectively purify the derivatized peptides from the sample,an affinity purification was carried out using an avidin column(Monomeric Avidin, Perbio Science Deutschland GmbH, Bonn) which wasprepared in-house. The column was prepared and used in accordance withthe manufacturer's instructions. The peptides were eluted with 0.4%trifluoroacetic acid/30% acetonitrile.

Mass-Spectrometric Analysis

An ion trap mass spectrometer (LCQdeka, ThermoFinnigan, San Jose) whichwas connected directly to a high pressure liquid chromatographyappliance (LC-MS) was used for analyzing the peptides. A reversed-phasecolumn (C₁₈ phase) was used as the separation column. The peptides weredissolved in eluent A (0.025% (v/v) trifluoroacetic acid) and injected.They were eluted with a gradient of eluent B (0.025% (v/v)trifluoroacetic acid/84% (v/v) acetonitrile). The eluting peptides wererecognized automatically by the acquisition software in the instrumentand fragmented for identification. In this way, it was possible todetermine the identities of the peptides unambiguously.

The following results were expected on the basis of the experimentalmixture:

-   -   all the peptides from the SDS-7 mixture were to be detected in        the form of four identically intensive signals which did not        show any isotope effect in the chromatography.    -   peptides from interleukin-4 were to appear in the form of a        doublet of signals having a mass difference of 17 Da.

FIG. 2 shows the ion traces for the four differently labeled variants ofthe peptide LQGIVSWGSGCAQK from Trypsinogen. From the top to the bottom,the traces relate to the peptide which has been labeled by the affinitytags with 0, 5, 11 and 17 isotope labels. It is not possible to measureany retention difference between variants. FIG. 3 shows the appurtenantMS spectrum, which contains a quadruplet of almost equally intensesignals. The identity of the peptide was confirmed by means of MS/MSexperiments.

FIG. 4 shows the signals of the peptide NLWGLAGLNSCPVK frominterleukin-4. As expected, a doublet of signals is seen, with theintensity ratio being 1:1. In this way, the signal can be distinguishedclearly from peptides which are unlabeled and which were purified bynonspecific adsorption on the affinity column. The identity of thepeptide was confirmed by means of MS/MS experiments.

FIG. 2: The ion traces for the m/z values 647.3 Da, 648.9 Da, 650.9 Daand 652.9 Da. The traces relate to the triply charged ion of the peptideLQGIVSWGSGCAQK in the forms in which it is reacted with Examples 38 to41. The identity of the peptides was confirmed by means of MS/MSexperiments.

FIG. 3: MS spectrum of the LC peaks shown in FIG. 2. The peptide ion istriply charged.

FIG. 4: MS spectrum of the triply charged ion of the peptideNLWGLAGLNSCPVK from interleukin-4. Since it was only present in sample1, a doublet of signals appears, with this doublet corresponding to thepeptide possessing 0 and, respectively, 17 isotope labels.

1. An organic compound of the formula (II),

in which the groups A, PRG, S, Z, L′, Z′ and k, l, m and n, are definedas follows: A is the acyl residue of an affinity ligand selected frombiotinyl and a biotin derivative, or is a functional group which isbound to a polymeric support, and is a support-bound hydroxyl, carboxylor amino group, PRG is the residue of a protein-reactive group, selectedfrom

 —CO—(CH₂)_(r)—Cl in which r=1–10, and  —CO—CH═CH₂; S is anacid-cleavable group of the formula

in which Y is a spacer selected from NH, NH—CH₂, NH—CH₂—CH₂—NH—CO andCH₂—CO, where Y can be in the ortho, meta or para position in relationto NH, and in which SK is the side chain residue of an α-amino acid ofthe formula SK—CH(NH₂)—COOH selected form the side chains of the 20natural amino acids, which, in the case of SKs other than an H atom, canbe present in the D, L or racemic from, Z is the residue of an aminoacid, which is not labeled or which can contain ¹³C or ¹⁵N labels, or acombination of these labels, selected from the 20 natural proteinogenicamino acids, and residues of ω-amino acids, selected from NH—(CH₂)₂—COand CO—(CH₂)₂—NH, which, where appropriate, can be in the D, L orracemic form, L′ is a bridge which makes possible, or facilitates, thecovalent linkage of two piperazine residues, which can, whereappropriate, contain ¹³C or ¹⁵N isotope labels or a combination of theselabels, selected from CO—CO, CO—(CH₂)_(s)—CO and also CO-arylene-CO,CO—CH₂—NH—CO—NH—CH₂—CO, CO—NH—CH₂—CO, CO—CH₂—NH—CO,CO—CH₂—NH—CO—CO—NH—CH₂—CO, CO—CH₂—NH—CO—(CH₂)_(s)—CO—NH—CH₂—CO,CO—CH₂—NH—CO-arylene-CO—NH—CH₂—CO, (CH₂)_(s)—NH—CO—CO—NH—(CH₂)_(s),(CH₂)₃—NH—CO—CO—NH—(CH₂)₃, (CH₂)_(s)—CO, (CH₂)₂—CO, CO and CS, where sis an integer between 1 and 6, R and R′ are hydrogen, Z′ is the residueof an amino acid, which differs from Z in the different orientation inregard to the terminal CO and NH groups; it may not be labeled or maycontain ¹³C or ¹⁵N labels, or a combination of these labels selectedfrom the 20 natural proteinogenic amino acids, and residues of ω-aminoacids, selected from NH—(CH₂)₂—CO and CO—(CH₂)₂—NH, which, whereappropriate, can be in the D, L or racemic form, k, l, m and n can,independently of each other, in each case be numbers between 0 and 10,where the sum of k+l+m+n is greater than 0 and less than 20, with theproviso that at least one of Z, L′, and Z′ is labeled with ¹³C and/or¹⁵N; or a pharmaceutically acceptable salt thereof.
 2. A compound asclaimed in claim 1, characterized in that Z is the residue of glycine.3. A compound as claimed in claim 1 or 2, characterized in that Z′ isthe residue of glycine.
 4. A compound as claimed in claim 1,characterized in that A is an amino-functionalized resin based onpolyethylene glycol or silica gel.
 5. A process for preparing a compoundas claimed in claim 1, in which  i) a protected intermediate of theformula (III)

 in which SG and SG′ are two orthogonal protecting groups, selected frombenzyloxycarbonyl, Boc, and Fmoc,  is prepared, ii) the protecting groupSG is first of all detached from the intermediate of the formula (III)and, after that, another amino acid derivative, which carries aprotecting group SG, which is identical to, or different from, thedetached protecting group, on the α-amino function, is attached, with aderivative of the formula (IV),

 in which SK is the side chain of an amino acid, as defined in claim 1, being obtained, iii) after the protecting group SG′ has been detachedfrom the derivative of the formula (IV), the latter is reacted with thederivative or the activated precursor of the derivative of the formula(V)U-PRG  (V)  in which U is a group which enables PRG to be linked to Z′or, where appropriate, to another end group of L, iv) the terminalprotecting group SG is detached, with a conjugate of the formula (VI)

 being obtained, v) an affinity ligand A-OH or A-NH₂, or ahydroxyl-functionalized, carboxyl-functionalized or amino-functionalizedsolid phase A-OH or A-NH₂, or an activated form thereof, is reacted witha compound of the formula (VII)

 in which Y is as defined in claim 1,  which can optionally carry aprotecting group selected from the group benzyloxycarbonyl, Boc, andFmoc, to give the derivative of the formula (VIII)

vi) the derivative of the formula (VIII) is then converted, after priorelimination of an optionally introduced protecting group, into acorresponding isothiocyanate, vii) the isothiocyanate is then coupled tothe conjugate of the formula (VI) to give the thiourea of the formula(IX), and

viii) in an optional last step, protecting groups which are stillpresent are eliminated, with it being possible to carry out theconsecutive steps v) and vi) at any arbitrary time prior to step vii).6. A process for preparing a compound as claimed in claim 1, in which i) a protected intermediate of the formula (III)

 in which SG and SG′ are two orthogonal protecting groups selected frombenzyloxycarbonyl, Boc, and Fmoc,  is prepared, ii) the protecting groupSG is first of all detached from the intermediate of the formula (III)and, after that, another amino acid derivative, which carries aprotecting group SG, which is identical to, or different from, thedetached protecting group, on the α-amino function, is attached, with aderivative of the formula (IV),

 in which SK is the side chain of an amino acid, as defined in claim 1, being obtained, iii) the terminal protecting group SG is detached, witha conjugate of the formula (VI′)

 being obtained, iv) an affinity ligand A-OH or A-NH₂, or ahydroxyl-functionalized, carboxyl-functionalized or amino-functionalizedsolid phase A-OH or A-NH₂, or an activated form thereof, is reacted witha compound of the formula (VII)

 in which Y is defined as in claim 1,  which can optionally carry aprotecting group, to give the derivative of the formula (VIII)

v) the derivative of the formula (VIII) is then converted, after theprior elimination of an optionally introduced protecting group, into acorresponding isothiocyanate, vi) the isothiocyanate is then coupled tothe conjugate of the formula (VI′) to give the thiourea of the formula(X′),

 and vii) after the protecting group SG′ has been detached from thethiourea of the formula (X′), the latter is reacted with the derivativeof a protein-reactive group or the activated precursor of the derivativeof the formula (V)U-PRG  (V)  in which U is a group which enables PRG to be linked to Z′or, where appropriate, to another end group of L,  and viii) in anoptional last step, protecting groups which may still be present areeliminated, with it being possible to carry out the consecutive stepsiv) and v) at any arbitrary time prior to step vi).
 7. A method for themass spectrometric analysis of proteins, comprising: a tagging proteinsin at least one mixture of proteins with at least one isotope-labeledcompound according to claim 1, b) if necessary, cleaving the taggedproteins to produce tagged peptides, c) purifying the tagged peptides byaffinity chromatography, and d) analyzing the tagged peptides by massspectrometry.
 8. The method of claim 7 wherein one or more of saidproteins is identified.
 9. The method of claim 7 wherein the relativelevel of expression of one or more proteins in one or moreprotein-containing samples is determined.
 10. A kit for themass-spectrometric analysis of proteins, comprising, as reagents, one ormore differently isotope-labeled compounds as claimed in claim 1.